Polysaccharide synthases (h)

ABSTRACT

The present invention relates generally to polysaccharide synthases. More particularly, the present invention relates to (1,3;1,4)-β- D -glucan synthases. The present invention provides, among other things, methods for influencing the level of (1,3;1,4)-β- D -glucan produced by a cell and nucleic acid and amino acid sequences which encode (1,3;1,4)-β- D -glucan synthases.

PRIORITY CLAIM

The present invention claims priority to Australian provisional patentapplication 2007907071 the content of which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates generally to polysaccharide synthases.More particularly, the present invention relates to (1,3;1,4)-β-D-glucansynthases.

BACKGROUND OF THE INVENTION

The various tissues of cereal grains have diverse functions during graindevelopment, dormancy and after germination.

For example, the pericarp and seed coat tissues are concerned with theprotection of the seed during development and during dormancy. However,by grain maturity, these outer grain tissues have died and the tissueresidues consist almost entirely of cell wall residues. The nucellartissue between the seed coat and the aleurone surface is involved intransfer of nutrients to the developing grain, however, at maturity thistissue has also collapsed to leave cell wall remnants. The thin walledcells of the starchy endosperm of mature grain are dead, but are packedwith starch and storage protein. In contrast, the thick-walled,nucleated, aleurone cells are alive at grain maturity, and are packedwith protein bodies and lipid droplets. At the interface of the starchyendosperm lies the scutellum, which functions in delivering nutrients tothe developing endosperm and, during germination, transfers digestionproducts of the endosperm reserves to the developing embryo.

The different structure and function of each tissue type in the grain isdetermined, at least in part, by the cell wall composition of each ofthese cell types.

Non-cellulosic polysaccharides are key components in the cell walls ofcereal grain tissues and include, for example, (1,3;1,4)-β-D-glucans,heteroxylans (mainly arabinoxylans), glucomannans, xyloglucans, pecticpolysaccharides and callose. These non-cellulosic polysaccharidesusually constitute less than 10% of the overall weight of the grain, butnevertheless are key determinants of grain quality.

Although the precise physical relationships between individualnon-cellulosic polysaccharides and other wall components have not beendescribed, it is generally considered that in the wall, microfibrils ofcellulose are embedded in a matrix phase of non-cellulosicpolysaccharides and protein. Wall integrity is maintained predominantlythrough extensive non-covalent interactions, especially hydrogenbonding, between the matrix phase and microfibrillar constituents. Inthe walls of some grain tissues covalent associations betweenheteroxylans, lignin and proteins are present. The extent of covalentassociations between components also varies with the wall type andgenotype.

Non-cellulosic polysaccharides, especially heteroxylans and(1,3;1,4)-β-D-glucans, constitute a relatively high proportion of thewalls of the aleurone and starchy endosperm, and probably also of thescutellum. In these tissues, cellulose contents are correspondinglylower. The generally low cellulose content of these walls, together withthe fact that they contain no lignin, are thought to be related to alimited requirement for structural rigidity of walls in central regionsof the grain, and to a requirement to rapidly depolymerize wallcomponents following germination of the grain.

In contrast, in the cell walls of the pericarp-seed coat, which providesa protective coat for the embryo and endosperm and which is notmobilized during germination, cellulose and lignin contents are muchhigher and the concentrations of non-cellulosic polysaccharides arecorrespondingly lower.

(1,3;1,4)-β-D-glucans, also referred to as mixed-linkage or cerealβ-glucans, are non-cellulosic polysaccharides which naturally occur inplants of the monocotyledon family Poaceae, to which the cereals andgrasses belong, and in related families of the order Poales.

These non-cellulosic polysaccharides are important constituents of thewalls of the starchy endosperm and aleurone cells of most cereal grains,where they can account for up to 70%-90% by weight of the cell walls.

Barley, oat and rye grains are rich sources of (1,3;1,4)-β-D-glucan,whereas wheat, rice and maize have lower concentrations of thispolysaccharide. The (1,3;1,4)-β-D-glucans are also relatively minorcomponents of walls in vegetative tissues of cereals and grasses.Although present as a relatively minor component in vegetative tissues(1,3;1,4)-β-D-glucan) is still important in terms of, for example, thedigestibility of vegetative tissue by animals and in the use of cropresidues for bioethanol production.

(1,3;1,4)-β-D-glucans are important in large-scale food processingactivities that include brewing and stockfeed manufacture. Moreover, thenon-starchy polysaccharides of cereals, such as (1,3;1,4)-β-D-glucans,have attracted renewed interest in recent years because of theirpotentially beneficial effects in human nutrition.

However, despite this interest, major gaps remain in our knowledge ofthe genes and enzymes that control non-cellulosic polysaccharidebiosynthesis, including (1,3;1,4)-β-D-glucan biosynthesis, in cerealgrain.

(1,3;1,4)-β-D-glucan concentrations in grain are thought to beinfluenced by both genotype and environment. For example, theconcentration of (1,3;1,4)-β-D-glucan in cereal grains depends on thegenotype, the position of the grain on the spike and environmentalfactors such as planting location, climatic conditions duringdevelopment and soil nitrogen.

Identification of the genes encoding (1,3;1,4)-β-D-glucan synthaseswould be desirable, as this would facilitate modulation of the level of(1,3;1,4)-β-D-glucan produced by a cell, and therefore, allow thequalities of grain or vegetative tissue to be altered. Therefore, inorder to enable the modulation of the level of (1,3;1,4)-β-D-glucan in acell and associated changes in grain or vegetative tissue quality, thereis a clear need to identify genes that encode (1,3;1,4)-β-D-glucansynthases.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

SUMMARY OF THE INVENTION

In accordance with the present invention, nucleotide sequences andcorresponding amino acid sequences that encode a family of(1,3;1,4)-β-D-glucan synthases are provided. In accordance with thepresent invention, it has been revealed that (1,3;1,4)-β-D-glucansynthases are encoded by members of the CslH gene family.

As a result of the identification of nucleotide sequences andcorresponding amino acid sequences that encode (1,3;1,4)-β-D-glucansynthases, the present invention provides, inter alia, methods andcompositions for modulating the level and/or activity of(1,3;1,4)-β-D-glucan synthase in a cell and/or modulating the level of(1,3;1,4)-β-D-glucan produced by the cell.

Therefore, in a first aspect, the present invention provides a methodfor modulating the level of (1,3;1,4)-β-D-glucan produced by a cell, themethod comprising modulating the level and/or activity of a CslH-encoded(1,3;1,4)-β-D-glucan synthase in the cell.

In some embodiments, the level and/or activity of a (1,3;1,4)-β-D-glucansynthase is modulated by modulating the expression of a CslH nucleicacid in the cell. Therefore, in a second aspect, the present inventionprovides a method for modulating the level and/or activity of a(1,3;1,4)-β-D-glucan synthase in a cell, the method comprisingmodulating the expression of a CslH nucleic acid in the cell.

In some embodiments, the present invention contemplates increasing thelevel of (1,3;1,4)-β-D-glucan produced by a cell, by expressing,overexpressing or introducing a CslH nucleic acid into the cell.Alternatively, in further embodiments the present invention alsoprovides methods for down-regulating expression of a CslH-encoded(1,3;1,4)-β-D-glucan synthase in a cell by knockout or knockdown of aCslH nucleic acid in a cell.

The present invention also facilitates the production of(1,3;1,4)-β-D-glucan in a recombinant expression system. For example,(1,3;1,4)-β-D-glucan may be recombinantly produced by introducing a CslHnucleic acid under the control of a promoter, into a cell, wherein thecell subsequently expresses a CslH-encoded (1,3;1,4)-β-D-glucan synthaseand produces (1,3;1,4)-β-D-glucan. Therefore, in a third aspect, thepresent invention provides a method for producing (1,3;1,4)-β-D-glucan,the method comprising transforming a cell with an isolated CslH nucleicacid and allowing the cell to express the isolated CslH nucleic acid.

In a fourth aspect, the present invention also provides(1,3;1,4)-β-D-glucan produced according to the method of the thirdaspect of the invention.

In a fifth aspect, the present invention also provides a cellcomprising:

-   -   a modulated level and/or activity of CslH-encoded        (1,3;1,4)-β-D-glucan synthase relative to a wild type cell of        the same taxon; and/or    -   modulated expression of a CslH nucleic acid relative to a wild        type cell of the same taxon.

In some embodiments, the cell further comprises a modulated level of(1,3;1,4)-β-D-glucan relative to a wild type cell of the same taxon.

Furthermore, in a sixth aspect, the present invention provides amulticellular structure comprising one or more cells according to thefifth aspect of the invention.

The present invention also provides cereal grain comprising one or morecells according to the fifth aspect of the invention. Therefore, in aseventh aspect, the present invention provides a cereal grain comprisinga modulated level of (1,3;1,4)-β-D-glucan, wherein the grain comprisesone or more cells comprising a modulated level and/or activity of aCslH-encoded (1,3;1,4)-β-D-glucan synthase and/or modulated expressionof a CslH nucleic acid.

In an eighth aspect, the present invention also provides flourcomprising:

-   -   flour produced by the milling of the grain of the seventh aspect        of the invention; and    -   optionally, flour produced by the milling of one or more other        grains.

As set out above, the present invention is predicated, in part, on theidentification and isolation of CslH nucleotide sequences and CslH aminoacid sequences that encode (1,3;1,4)-β-D-glucan synthases.

Therefore, in a ninth aspect, the present invention provides an isolatedCslH nucleic acid or a complement, reverse complement or fragmentthereof.

In a tenth aspect, the present invention provides a genetic construct orvector comprising an isolated nucleic acid molecule of the ninth aspectof the invention.

In an eleventh aspect, the present invention provides a cell comprisingthe isolated nucleic acid molecule of the ninth aspect of the inventionor genetic construct of the tenth aspect of the invention.

In a twelfth aspect, the present invention provides a multicellularstructure comprising one or more of the cells of the eleventh aspect ofthe invention.

As set out above, the present invention also provides amino acidsequences for CslH-encoded (1,3;1,4)-β-D-glucan synthases.

Accordingly, in a thirteenth aspect, the present invention provides anisolated polypeptide comprising an amino acid sequence defining aCslH-encoded (1,3;1,4)-β-D-glucan synthase polypeptide or a fragmentthereof.

In a fourteenth aspect, the present invention provides an antibody or anepitope binding fragment thereof, raised against an isolatedCslH-encoded (1,3;1,4)-β-D-glucan synthase polypeptide as hereinbeforedefined or an epitope thereof.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to herein by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO: 1), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided at the end of the specification.

TABLE 1 Summary of Sequence Identifiers Sequence Identifier Sequence SEQID NO: 1 HvCslH1 coding region nucleotide sequence SEQ ID NO: 2 HvCslH1amino acid sequence SEQ ID NO: 3 OsCslH1 coding region nucleotidesequence SEQ ID NO: 4 OsCslH1 amino acid sequence SEQ ID NO: 5 OsCslH2coding region nucleotide sequence SEQ ID NO: 6 OsCslH2 amino acidsequence SEQ ID NO: 7 OsCslH3 coding region nucleotide sequence SEQ IDNO: 8 OsCslH3 amino acid sequence SEQ ID NO: 9 HvCslH1 genomicnucleotide sequence SEQ ID NO: 10 OsCslH1 genomic nucleotide sequenceSEQ ID NO: 11 OsCslH2 genomic nucleotide sequence SEQ ID NO: 12 OsCslH3genomic nucleotide sequence SEQ ID NO: 13 H1F1 primer nucleotidesequence SEQ ID NO: 14 H1F2 primer nucleotide sequence SEQ ID NO: 15HvCslH1cF1 primer nucleotide sequence SEQ ID NO: 16 HvH1TOPOf primernucleotide sequence SEQ ID NO: 17 H1R1 primer nucleotide sequence SEQ IDNO: 18 H1R2 primer nucleotide sequence SEQ ID NO: 19 H1R5 primernucleotide sequence SEQ ID NO: 20 H1R6 primer nucleotide sequence SEQ IDNO: 21 H1R7 primer nucleotide sequence SEQ ID NO: 22 H1R10 primernucleotide sequence SEQ ID NO: 23 HvCslH1cR1 primer nucleotide sequenceSEQ ID NO: 24 HvH1TOPOr primer nucleotide sequence SEQ ID NO: 25 Adaptor1 primer nucleotide sequence SEQ ID NO: 26 Adaptor 2 primer nucleotidesequence SEQ ID NO: 27 AP1 primer nucleotide sequence SEQ ID NO: 28 AP2primer nucleotide sequence SEQ ID NO: 29 Hv18SRTr primer nucleotidesequence SEQ ID NO: 30 Hv18Sf primer nucleotide sequence SEQ ID NO: 31Hv18Sr primer nucleotide sequence SEQ ID NO: 32 Hv GAPDH Q-PCR forwardprimer nucleotide sequence SEQ ID NO: 33 Hv GAPDH Q-PCR reverse primernucleotide sequence SEQ ID NO: 34 Hv Cyclophilin Q-PCR forward primernucleotide sequence SEQ ID NO: 35 Hv Cyclophilin Q-PCR reverse primernucleotide sequence SEQ ID NO: 36 Hv α-Tubulin Q-PCR forward primernucleotide sequence SEQ ID NO: 37 Hv α-Tubulin Q-PCR reverse primernucleotide sequence SEQ ID NO: 38 Hv HSP70 Q-PCR forward primernucleotide sequence SEQ ID NO: 39 Hv HSP70 Q-PCR reverse primernucleotide sequence SEQ ID NO: 40 Hv EL1a Q-PCR forward primernucleotide sequence SEQ ID NO: 41 Hv EL1a Q-PCR reverse primernucleotide sequence SEQ ID NO: 42 HvCslH1 Q-PCR forward primernucleotide sequence SEQ ID NO: 43 HvCslH1 Q-PCR reverse primernucleotide sequence SEQ ID NO: 44 SJ27 primer nucleotide sequence SEQ IDNO: 45 SJ28 primer nucleotide sequence SEQ ID NO: 46 SJ72 primernucleotide sequence SEQ ID NO: 47 SJ73 primer nucleotide sequence SEQ IDNO: 48 SJ79 primer nucleotide sequence SEQ ID NO: 49 SJ75 primernucleotide sequence SEQ ID NO: 50 SJ85 primer nucleotide sequence SEQ IDNO: 51 SJ91 primer nucleotide sequence SEQ ID NO: 52 SJ163 primernucleotide sequence SEQ ID NO: 53 SJ164 primer nucleotide sequence SEQID NO: 54 SJ183 primer nucleotide sequence SEQ ID NO: 55 SJ204 primernucleotide sequence SEQ ID NO: 56 TUB primer nucleotide sequence SEQ IDNO: 57 TUB2F primer nucleotide sequence SEQ ID NO: 58 SJ107 primernucleotide sequence SEQ ID NO: 59 SJ82 primer nucleotide sequence SEQ IDNO: 60 SJ94 primer nucleotide sequence SEQ ID NO: 61 SJ95 primernucleotide sequence SEQ ID NO: 62 SJ97 primer nucleotide sequence SEQ IDNO: 63 SJ93 primer nucleotide sequence SEQ ID NO: 64 SJ44 primernucleotide sequence SEQ ID NO: 65 SJ38 primer nucleotide sequence SEQ IDNO: 66 SJ96 primer nucleotide sequence SEQ ID NO: 67 SJ37 primernucleotide sequence SEQ ID NO: 68 SJ244 primer nucleotide sequence SEQID NO: 69 HvCslH1 (cv. Himalaya) coding region nucleotide sequence SEQID NO: 70 HvCslH1 (cv. Himalaya) amino acid sequence SEQ ID NO: 71HvCslH1 (cv. Himalaya) genomic nucleotide sequence SEQ ID NO: 72TaCslH1-1 coding region nucleotide sequence SEQ ID NO: 73 TaCslH1-2coding region nucleotide sequence SEQ ID NO: 74 TaCslH1-3 coding regionnucleotide sequence SEQ ID NO: 75 TaCslH1-1 amino acid sequence SEQ IDNO: 76 TaCslH1-2 amino acid sequence SEQ ID NO: 77 TaCslH1-3 amino acidsequence SEQ ID NO: 78 TaCslH1-1 genomic nucleotide sequence SEQ ID NO:79 TaCslH1-2 genomic nucleotide sequence SEQ ID NO: 80 TaCslH1-3 genomicnucleotide sequence

DESCRIPTION OF EXEMPLARY EMBODIMENTS

It is to be understood that following description is for the purpose ofdescribing particular embodiments only and is not intended to belimiting with respect to the above description.

The present invention is predicated, in part, on the identification ofgenes which encode biosynthetic enzymes for (1,3;1,4)-β-D-glucans.

“(1,3;1,4)-β-D-glucans” should be understood to include linear,unbranched polysaccharides in which β-D-glucopyranosyl monomers arepolymerized through both (1→4)- and (1→3)-linkages.

The ratio of (1→4)- to (1→3)-linkages in naturally occurring(1,3;1,4)-β-D-glucans is generally in the range 2.2-2.6:1, although theratio may also be outside of this range. For example, in(1,3;1,4)-β-D-glucan from sorghum endosperm the ratio is 1.15:1. The twotypes of linkages are not arranged in regular, repeating sequences.Single (1→3)-linkages are separated by two or more (1→4)-linkages.Regions of two or three adjacent (1→4)-linkages predominate, but againthere is no regularity in the arrangement of these units. The linkagesequence does not depend on preceding linkages further away than twoglucose units and follows a second order Markov chain distribution.Moreover, up to 10% of the chain may consist of longer stretches of 5 to20 adjacent (1→4)-linkages. Thus, cereal (1,3;1,4)-β-D-glucans may beconsidered as (1→3)-β-linked copolymers of cellotriosyl (G4G4G_(Red)),cellotetraosyl (G4G4G4G_(Red)) units and longer (1→4)-β-D-oligoglucosylunits.

The ratio of tri- to tetra-saccharide units in endogenous(1,3;1,4)-β-D-glucans varies between cereal species. For example, inwheat the ratio is 3.0-4.5:1, in barley 2.9-3.4:1, in rye 2.7:1 and inoats 1.8-2.3:1. Furthermore, the observed ratios may also vary accordingto the temperature and conditions of (1,3;1,4)-β-D-glucan extraction.

The average molecular masses reported for cereal (1,3;1,4)-β-D-glucansrange from 48,000 (DP ˜300) to 3,000,000 (DP ˜1850), depending on thecereal species, cell wall type, extraction procedure and the method usedfor molecular mass determination. They are invariably polydisperse withrespect to molecular mass and this is illustrated by a weight average tonumber average molecular mass ratio (M_(w)/M_(n)) of 1.18 for barley(1,3;1,4)-β-D-glucan. Certain barley (1,3;1,4)-β-D-glucans are alsocovalently-associated with small amounts of protein and have estimatedmolecular masses of up to 40,000,000.

The extractability of (1,3;1,4)-β-D-glucans from walls of cereal grainsis a function of their degree of self-association and their associationwith other wall polysaccharides and proteins. In particular,extractability depends on the molecular mass and linkage distribution inthe (1,3;1,4)-β-D-glucan chains. Extensive association with otherpolymers and very high molecular masses render the (1,3;1,4)-β-D-glucansmore difficult to extract from grain.

For example, a portion of the (1,3;1,4)-β-D-glucan from barley, oat andrye flours may be extracted by water at pH 7.0 and 40° C. Furtherfractions can be solubilised at higher temperatures. The proportion oftotal (1,3;1,4)-β-D-glucan that is water-soluble at 40° C. varies withinand between species. For example, waxy (high amylose) barleys have ahigher proportion of water-soluble (1,3;1,4)-β-D-glucan than normalbarleys. (1,3;1,4)-β-D-glucans extracted from barley at 40° C. have aslightly lower tri-/tetrasaccharide ratio (1.7:1) than those extractedat 65° C. (2.0:1). Complete extraction of cereal (1,3;1,4)-β-D-glucansfrom grain requires the use of alkaline extractants such as 4 M NaOH oraqueous Ba(OH)₂, containing NaBH₄ to prevent alkali-induced degradationfrom the reducing terminus. Alkali-extracted barley (1,3;1,4)-β-D-glucanfractions have higher molecular masses, higher ratios of (1→4):(1→3)linkages, more contiguously linked (1→4)-linked segments and highertri-:tetra-saccharide ratios than their water-extractable counterparts.Other extractants, such as dimethylsulphoxide, hot perchloric acid,trichloroacetic acid, N-methylmorpholino-N-oxide anddimethylacetamide-LiCl, may also be used to solubilise(1,3;1,4)-β-D-glucans, but these extractants may cause somedepolymerisation or degradation of the polymer. Once extracted with hotwater or alkali, the (1,3;1,4)-β-D-glucans are often soluble at neutralpH and room temperature. However, upon cooling, (1,3;1,4)-β-D-glucanscan aggregate and precipitate.

As mentioned above, the present invention is predicated, in part, on theidentification of biosynthetic enzymes, and their encoding genes, thatcatalyse the synthesis of (1,3;1,4)-β-D-glucan. Such enzymes arereferred to herein as “(1,3;1,4)-β-D-glucan synthases”.

The present invention arises, in part, from an analysis of expressedsequence tag libraries and other sequence databases including cellulosesynthase (CesA) genes. More particularly, it was noted in these analysesthat the CesA genes were in fact members of a much larger super-familyof genes, which included both the CesA genes and the cellulosesynthase-like (Csl) gene family.

The Csl gene families in most vascular plants are very large and havebeen divided into several groups, designated CslA to CslH. InArabidopsis thaliana there are 29 known Csl genes and in rice about 37.Overall, the Arabidopsis genome is believed to contain more than 700genes involved in cell wall metabolism. However, in general, thespecific functions of these genes are poorly understood.

Furthermore, in contrast to the CesA genes, it has proved difficult todefine the functions of the Csl genes. In fact, of the multiple Cslgenes in higher plants, only the CslA and CslF groups have been assigneda function.

In accordance with the present invention, it has been revealed thatmembers of the CslH gene family encode (1,3;1,4)-β-D-glucan synthases.

As a result of the identification of CslH nucleotide sequences, andcorresponding amino acid sequences that encode (1,3;1,4)-β-D-glucansynthases, the present invention provides, inter alia, methods andcompositions for modulating the level and/or activity of(1,3;1,4)-β-D-glucan synthase in a cell and/or modulating the level of(1,3;1,4)-β-D-glucan produced by the cell.

Therefore, in a first aspect, the present invention provides a methodfor modulating the level of (1,3;1,4)-β-D-glucan produced by a cell, themethod comprising modulating the level and/or activity of a CslH-encoded(1,3;1,4)-β-D-glucan synthase in the cell.

The “cell” may be any suitable eukaryotic or prokaryotic cell. As such,a “cell” as referred to herein may be a eukaryotic cell including afungal cell such as a yeast cell or mycelial fungus cell; an animal cellsuch as a mammalian cell or an insect cell; or a plant cell.Alternatively, the cell may also be a prokaryotic cell such as abacterial cell including an E. coli cell, or an archaea cell.

In some embodiments, the cell is a plant cell, a vascular plant cell,including a monocotyledonous or dicotyledonous angiosperm plant cell, ora gymnosperm plant cell. In some embodiments the plant is amonocotyledonous plant cell. In some embodiments, the plant is a memberof the order Poales. In some embodiments, the monocotyledonous plantcell is a cereal crop plant cell.

As used herein, the term “cereal crop plant” includes members of thePoales (grass family) that produce edible grain for human or animalfood. Examples of Poales cereal crop plants which in no way limit thepresent invention include wheat, rice, maize, millet, sorghum, rye,triticale, oats, barley, teff, wild rice, spelt and the like. However,the term cereal crop plant should also be understood to include a numberof non-Poales species that also produce edible grain and are known asthe pseudocereals, such as amaranth, buckwheat and quinoa.

In other embodiments, the present invention also contemplates the use ofother monocotyledonous plants, such as other non-cereal plants of thePoales, specifically including pasture grasses such as Lolium spp.

As set out above, the present invention is predicated, in part, onmodulating the level and/or activity of a CslH-encoded(1,3;1,4)-β-D-glucan synthase in a cell.

A “CslH-encoded (1,3;1,4)-β-D-glucan synthase” should be regarded as anyCslH-encoded protein which catalyses the synthesis of(1,3;1,4)-β-D-glucan and, optionally, catalyses the polymerisation ofglucopyranosyl monomers.

In some embodiments, the CslH-encoded (1,3;1,4)-β-D-glucan synthasecomprises the amino acid sequence set forth in SEQ ID NO: 2 or an aminoacid sequence which is at least 50% identical thereto.

In some embodiments the CslH-encoded (1,3;1,4)-β-D-glucan synthasecomprises at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%,at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, atleast 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%,at least 99% at least 99.5% or 100% amino acid sequence identity to SEQID NO: 2.

When comparing amino acid sequences, the compared sequences should becompared over a comparison window of at least 100 amino acid residues,at least 200 amino acid residues, at least 400 amino acid residues, atleast 800 amino acid residues or over the full length of SEQ ID NO: 2.The comparison window may comprise additions or deletions (i.e. gaps) ofabout 20% or less as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by computerized implementations of algorithmssuch the BLAST family of programs as, for example, disclosed by Altschulet al. (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion ofsequence analysis can be found in Unit 19. 3 of Ausubel et al. (CurrentProtocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998,Chapter 15, 1998).

Examples of additional CslH-encoded (1,3;1,4)-β-D-glucan synthasescontemplated by the present invention include CslH-encoded(1,3;1,4)-β-D-glucan synthase orthologs of SEQ ID NO: 2.

For example, barley (Hordeum vulgare) orthologs or allelic variants ofSEQ ID NO: 2 include, for example, polypeptides comprising the aminoacid sequence set forth in SEQ ID NO: 70. Rice (Oryza sativa) orthologsof SEQ ID NO: 2 include, for example, polypeptides comprising the aminoacid sequences set forth in any of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ IDNO: 8. Wheat (Triticum aestivum) orthologs of SEQ ID NO: 2 include, forexample, polypeptides comprising the amino acid sequences set forth inSEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77.

As referred to herein, modulation of the “level” of the CslH-encoded(1,3;1,4)-β-D-glucan synthase should be understood to include modulationof the level of CslH transcripts and/or CslH-encoded(1,3;1,4)-β-D-glucan synthase polypeptides in the cell. Modulation ofthe “activity” of the CslH-encoded (1,3;1,4)-β-D-glucan synthase shouldbe understood to include modulation of the total activity, specificactivity, half-life and/or stability of the CslH-encoded(1,3;1,4)-β-D-glucan synthase in the cell.

By “modulating” with regard to the level and/or activity of theCslH-encoded (1,3;1,4)-β-D-glucan synthase is intended decreasing orincreasing the level and/or activity of CslH-encoded(1,3;1,4)-β-D-glucan synthase in the cell. By “decreasing” is intended,for example, a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% reduction in the level oractivity of CslH-encoded (1,3;1,4)-β-D-glucan synthase in the cell. By“increasing” is intended, for example, a 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20 fold,50-fold, 100-fold increase in the level of activity of CslH-encoded(1,3;1,4)-β-D-glucan synthase in the cell. “Modulating” also includesintroducing a CslH-encoded (1,3;1,4)-β-D-glucan synthase into a cellwhich does not normally express the introduced enzyme, or thesubstantially complete inhibition of CslH-encoded (1,3;1,4)-β-D-glucansynthase activity in a cell that normally has such activity.

In some embodiments, the level of (1,3;1,4)-β-D-glucan produced by acell is increased by increasing the level and/or activity ofCslH-encoded (1,3;1,4)-β-D-glucan synthase in the cell. In anotherembodiment, the level of (1,3;1,4)-β-D-glucan produced by a cell isdecreased by decreasing the level and/or activity of CslH-encoded(1,3;1,4)-β-D-glucan synthase in the cell.

The methods of the present invention contemplate any means known in theart by which the level and/or activity of a CslH-encoded(1,3;1,4)-β-D-glucan synthase in a cell may be modulated. This includesmethods such as the application of agents which modulate CslH-encoded(1,3;1,4)-β-D-glucan synthase activity in a cell, such as theapplication of a CslH-encoded (1,3;1,4)-β-D-glucan synthase agonist orantagonist; the application of agents which mimic CslH-encoded(1,3;1,4)-β-D-glucan synthase activity in a cell; modulating theexpression of a CslH nucleic acid which encodes CslH-encoded(1,3;1,4)-β-D-glucan synthase in the cell; or effecting the expressionof an altered or mutated CslH nucleic acid in a cell such that a(1,3;1,4)-β-D-glucan synthase with increased or decreased specificactivity, half-life and/or stability is expressed by the cell.

In some embodiments, the level and/or activity of a (1,3;1,4)-β-D-glucansynthase is modulated by modulating the expression of a CslH nucleicacid in the cell.

Therefore, in a second aspect, the present invention provides a methodfor modulating the level and/or activity of a (1,3;1,4)-β-D-glucansynthase in a cell, the method comprising modulating the expression of aCslH nucleic acid in the cell.

As used herein, the term “CslH nucleic acid” should be understood toinclude to a nucleic acid molecule which:

-   -   encodes a CslH-encoded (1,3;1,4)-β-D-glucan synthase as defined        herein; and/or    -   comprises at least 50% nucleotide sequence identity to the        nucleotide sequence set forth in SEQ ID NO: 1; and/or    -   hybridises to a nucleic acid molecule comprising one or more of        the nucleotide sequence set forth in SEQ ID NO: 1 under        stringent conditions.

In some embodiments the CslH nucleic acid comprises at least 50%, atleast 51%, at least 52%, at least 53%, at least 54%, at least 55%, atleast 56%, at least 57%, at least 58%, at least 59%, at least 60%, atleast 61%, at least 62%, at least 63%, at least 64%, at least 65%, atleast 66%, at least 67%, at least 68%, at least 69%, at least 70%, atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%,at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, atleast 97.5%, at least 98%, at least 98.5%, at least 99% at least 99.5%or 100% sequence identity to SEQ ID NO: 1.

When comparing nucleic acid sequences to SEQ ID NO: 1 to calculate apercentage identity, the compared nucleotide sequences should becompared over a comparison window of at least 300 nucleotide residues,at least 600 nucleotide residues, at least 1200 nucleotide residues, atleast 2400 nucleotide residues or over the full length of SEQ ID NO: 1.The comparison window may comprise additions or deletions (ie. gaps) ofabout 20% or less as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by computerized implementations of algorithmssuch the BLAST family of programs as, for example, disclosed by Altschulet al. (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion ofsequence analysis can be found in Unit 19. 3 of Ausubel et al. (“CurrentProtocols in Molecular Biology” John Wiley & Sons Inc, 1994-1998,Chapter 15, 1998).

As set out above, the CslH nucleic acid may also comprise a nucleic acidthat hybridises to a nucleic acid molecule comprising the nucleotidesequence set forth in SEQ ID NO: 1 under stringent conditions. As usedherein, “stringent” hybridisation conditions will be those in which thesalt concentration is less than about 1.5 M Na ion, typically about 0.01to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least 30° C. Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide. Stringenthybridisation conditions may be low stringency conditions, mediumstringency conditions or high stringency conditions. Exemplary lowstringency conditions include hybridisation with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary medium stringency conditions includehybridisation in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridisation in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity of hybridisation is also a function of post-hybridizationwashes, and is influenced by the ionic strength and temperature of thefinal wash solution. For DNA-DNA hybrids, the T_(m) can be approximatedfrom the equation of Meinkoth and Wahl (Anal. Biochem. 138: 267-284,1984), ie. T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L;where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of differentdegrees of complementarity. For example, sequences with ≧90% identitycan be hybridised by decreasing the T_(m) by about 10° C. Generally,stringent conditions are selected to be lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. For example, high stringency conditions canutilize a hybridization and/or wash at, for example, 1, 2, 3, 4 or 5° C.lower than the thermal melting point (T_(m)); medium stringencyconditions can utilize a hybridization and/or wash at, for example, 6,7, 8, 9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at, forexample, 11, 12, 13, 14, 15, or 20° C. lower than the thermal meltingpoint (T_(m)). Using the equation, hybridization and wash compositions,and desired T_(m), those of ordinary skill will understand thatvariations in the stringency of hybridization and/or wash solutions areinherently described. If the desired degree of mismatching results in aT_(m) of less than 45° C. (aqueous solution) or 32° C. (formamidesolution), the SSC concentration may be increased so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (Laboratory Techniques in Biochemistryand Molecular Biology-Hybridization with Nucleic Acid Probes, Pt I,Chapter 2, Elsevier, New York, 1993), Ausubel et al., eds. (CurrentProtocols in Molecular Biology, Chapter 2, Greene Publishing andWiley-Interscience, New York, 1995) and Sambrook et al. (MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y., 1989).

Examples of additional CslH nucleic acids contemplated by the presentinvention include nucleic acids having coding regions which areorthologs of SEQ ID NO: 1.

For example, barley (Hordeum vulgare) coding region orthologs or allelicvariants of SEQ ID NO: 1 include, for example, nucleic acids comprisingthe nucleotide sequence set forth in SEQ ID NO: 69. Rice (Oryza sativa)coding region orthologs of SEQ ID NO: 1 include, for example, nucleicacids comprising the nucleotide sequence set forth in any of SEQ ID NO:3, SEQ ID NO: 5 and SEQ ID NO: 7. Wheat (Triticum aestivum) codingregion orthologs of SEQ ID NO: 1 include, for example, nucleic acidscomprising the nucleotide sequence set forth in SEQ ID NO: 72, SEQ IDNO: 73 and SEQ ID NO: 74.

The CslH nucleic acids contemplated by the present invention may alsocomprise one or more non-translated regions such as 3′ and 5′untranslated regions and/or introns. For example, the CslH nucleic acidscontemplated by the present invention may comprise, for example, mRNAsequences, cDNA sequences or genomic nucleotide sequences

In some specific embodiments, the CslH nucleic acid may comprise agenomic nucleotide sequence from an organism which may include one ormore non-protein-coding regions and/or one or more introns. Genomicnucleotide sequences which comprise a CslH nucleic acid include, forexample:

-   -   barley (Hordeum vulgare) CslH genomic nucleotide sequences, for        example, as set forth in SEQ ID NO: 9 and/or SEQ ID NO: 71;    -   rice (Oryza sativa) CslH genomic nucleotide sequences, for        example, as set forth in any one or more of SEQ ID NO: 10, SEQ        ID NO: 11 and/or SEQ ID NO: 12; and/or    -   wheat (Triticum aestivum) CslH genomic nucleotide sequences, for        example, as set forth in any one or more of SEQ ID NO: 78, SEQ        ID NO: 79 and/or SEQ ID NO: 80.

As mentioned above, the present invention provides methods formodulating the expression of a CslH nucleic acid in a cell. The presentinvention contemplates any method by which the expression of a CslHnucleic acid in a cell may be modulated.

The term “modulating” with regard to the expression of the CslH nucleicacid is generally intended to refer to decreasing or increasing thetranscription and/or translation of a CslH nucleic acid. By “decreasing”is intended, for example, a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% reduction inthe transcription and/or translation of a CslH nucleic acid. By“increasing” is intended, for example a 1%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold,100-fold or greater increase in the transcription and/or translation ofa CslH nucleic acid. Modulating also comprises introducing expression ofa CslH nucleic acid not normally found in a particular cell; or thesubstantially complete inhibition (eg. knockout) of expression of a CslHnucleic acid in a cell that normally has such activity.

Methods for modulating the expression of a particular nucleic acidmolecule in a cell are known in the art and the present inventioncontemplates any such method. Exemplary methods for modulating theexpression of a CslH nucleic acid include: genetic modification of thecell to upregulate or downregulate endogenous CslH nucleic acidexpression; genetic modification by transformation with a CslH nucleicacid; administration of a nucleic acid molecule to the cell whichmodulates expression of an endogenous CslH nucleic acid in the cell; andthe like.

In some embodiments, the expression of a CslH nucleic acid is modulatedby genetic modification of the cell. The term “genetically modified”, asused herein, should be understood to include any genetic modificationthat effects an alteration in the expression of a CslH nucleic acid inthe genetically modified cell relative to a non-genetically modifiedform of the cell. Exemplary types of genetic modification contemplatedherein include: random mutagenesis such as transposon, chemical, UV orphage mutagenesis together with selection of mutants which overexpressor underexpress an endogenous CslH nucleic acid; transient or stableintroduction of one or more nucleic acid molecules into a cell whichdirect the expression and/or overexpression of CslH nucleic acid in thecell; site-directed mutagenesis of an endogenous CslH nucleic acid;introduction of one or more nucleic acid molecules which inhibit theexpression of an endogenous CslH nucleic acid in the cell, eg. acosuppression construct or an RNAi construct; and the like.

In one particular embodiment, the genetic modification comprises theintroduction of a nucleic acid into a cell of interest.

The nucleic acid may be introduced using any method known in the artwhich is suitable for the cell type being used, for example, thosedescribed in Sambrook and Russell (Molecular Cloning—A LaboratoryManual, 3^(rd) Ed., Cold Spring Harbor Laboratory Press, 2000).

In some embodiments of the invention wherein the cell is a plant cell,suitable methods for introduction of a nucleic acid molecule mayinclude: Agrobacterium-mediated transformation, microprojectilebombardment based transformation methods and direct DNA uptake basedmethods. Roa-Rodriguez et al. (Agrobacterium-mediated transformation ofplants, 3^(rd) Ed. CAMBIA Intellectual Property Resource, Can berra,Australia, 2003) review a wide array of suitable Agrobacterium-mediatedplant transformation methods for a wide range of plant species.Microprojectile bombardment may also be used to transform plant tissueand methods for the transformation of plants, particularly cerealplants, and such methods are reviewed by Casas et al. (Plant BreedingRev. 13: 235-264, 1995). Direct DNA uptake transformation protocols suchas protoplast transformation and electroporation are described in detailin Galbraith et al. (eds.), Methods in Cell Biology Vol. 50, AcademicPress, San Diego, 1995). In addition to the methods mentioned above, arange of other transformation protocols may also be used. These includeinfiltration, electroporation of cells and tissues, electroporation ofembryos, microinjection, pollen-tube pathway, silicon carbide- andliposome mediated transformation. Methods such as these are reviewed byRakoczy-Trojanowska (Cell. Mol. Biol. Lett. 7: 849-858, 2002). A rangeof other plant transformation methods may also be evident to those ofskill in the art.

The introduced nucleic acid may be single stranded or double stranded.The nucleic acid may be transcribed into mRNA and translated into aCslH-encoded (1,3;1,4)-β-D-glucan synthase or another protein; mayencode for a non-translated RNA such as an RNAi construct, cosuppressionconstruct, antisense RNA, tRNA, miRNA, siRNA, ntRNA and the like; or mayact directly in the cell. The introduced nucleic acid may be anunmodified DNA or RNA or a modified DNA or RNA which may includemodifications to the nucleotide bases, sugar or phosphate backbones butwhich retain functional equivalency to a nucleic acid. The introducednucleic acid may optionally be replicated in the cell; integrated into achromosome or any extrachromosomal elements of the cell; and/ortranscribed by the cell. Also, the introduced nucleic acid may be eitherhomologous or heterologous with respect to the host cell. That is, theintroduced nucleic acid may be derived from a cell of the same speciesas the genetically modified cell (ie. homologous) or the introducednucleic may be derived from a different species (ie. heterologous). Thetransgene may also be a synthetic transgene.

In one particular embodiment, the present invention contemplatesincreasing the level of (1,3;1,4)-β-D-glucan produced by a cell, byexpressing, overexpressing or introducing a CslH nucleic acid into thecell.

By identifying CslH nucleotide sequences which encode(1,3;1,4)-β-D-glucan synthases, in further embodiments, the presentinvention also provides methods for down-regulating expression of aCslH-encoded (1,3;1,4)-β-D-glucan synthase in a cell.

For example, the identification of CslH genes as encoding(1,3;1,4)-β-D-glucan synthases facilitates methods such as knockout orknockdown of a CslH-encoded (1,3;1,4)-β-D-glucan synthase in a cellusing methods such as:

-   -   insertional mutagenesis of a CslH nucleic acid in a cell,        including knockout or knockdown of a CslH nucleic acid in a        cell, by homologous recombination with a knockout construct (for        an example of targeted gene disruption in plants see Terada et        al., Nat. Biotechnol. 20: 1030-1034, 2002);    -   post-transcriptional gene silencing (PTGS) or RNAi of a CslH        nucleic acid in a cell (for review of PTGS and RNAi see Sharp,        Genes Dev. 15(5): 485-490, 2001; and Hannon, Nature 418: 244-51,        2002);    -   transformation of a cell with an antisense construct directed        against a CslH nucleic acid (for examples of antisense        suppression in plants see van der Krol et al., Nature 333:        866-869; van der Krol et al., Bio Techniques 6: 958-967; and van        der Krol et al., Gen. Genet. 220: 204-212);    -   transformation of a cell with a co-suppression construct        directed against a CslH nucleic acid (for an example of        co-suppression in plants see van der Krol et al., Plant Cell        2(4): 291-299);    -   transformation of a cell with a construct encoding a double        stranded RNA directed against a CslH nucleic acid (for an        example of dsRNA mediated gene silencing see Waterhouse et al.,        Proc. Natl. Acad. Sci. USA 95: 13959-13964, 1998);    -   transformation of a cell with a construct encoding an siRNA or        hairpin RNA directed against a CslH nucleic acid (for an example        of siRNA or hairpin RNA mediated gene silencing in plants see Lu        et al., Nucl. Acids Res. 32(21): e171; doi:10.1093/nar/gnh170,        2004); and/or    -   insertion of a miRNA target sequence such that it is in operable        connection with CslH nucleic acid (for an example of miRNA        mediated gene silencing see Brown et al., Blood 110(13):        4144-4152, 2007).

The present invention also facilitates the downregulation of a CslHnucleic acid in a cell via the use of synthetic oligonucleotides such assiRNAs or microRNAs directed against a CslH nucleic acid which areadministered to a cell (for examples of synthetic siRNA mediatedsilencing see Caplen et al., Proc. Natl. Acad. Sci. USA 98: 9742-9747,2001; Elbashir et al., Genes Dev. 15: 188-200, 2001; Elbashir et al.,Nature 411: 494-498, 2001; Elbashir et al., EMBO J. 20: 6877-6888, 2001;and Elbashir et al., Methods 26: 199-213, 2002).

In addition to the examples above, the introduced nucleic acid may alsocomprise a nucleotide sequence which is not directly related to a CslHnucleic acid but, nonetheless, may directly or indirectly modulate theexpression of CslH nucleic acid in a cell. Examples include nucleic acidmolecules that encode transcription factors or other proteins whichpromote or suppress the expression of an endogenous CslH nucleic acidmolecule in a cell; and other non-translated RNAs which directly orindirectly promote or suppress endogenous CslH-encoded(1,3;1,4)-β-D-glucan synthase expression and the like.

In order to effect expression of an introduced nucleic acid in agenetically modified cell, where appropriate, the introduced nucleicacid may be operably connected to one or more control sequences. Theterm “control sequences” should be understood to include any nucleotidesequences which are necessary or advantageous for the transcription,translation and or post-translational modification of the operablyconnected nucleic acid or the transcript or protein encoded thereby.Each control sequence may be native or foreign to the operably connectednucleic acid. The control sequences may include, but are not limited to,a leader, polyadenylation sequence, propeptide encoding sequence,promoter, enhancer or upstream activating sequence, signal peptideencoding sequence, and transcription terminator. Typically, a controlsequence at least includes a promoter.

The term “promoter” as used herein, describes any nucleic acid whichconfers, activates or enhances expression of a nucleic acid molecule ina cell. Promoters are generally positioned 5′ (upstream) to the genesthat they control. In the construction of heterologous promoter/genecombinations, it may be desirable to position the promoter at a distancefrom the gene transcription start site that is approximately the same asthe distance between that promoter and the gene it controls in itsnatural setting, ie. the gene from which the promoter is derived. As isknown in the art, some variation in this distance can be accommodatedwithout loss of promoter function.

A promoter may regulate the expression of an operably connectednucleotide sequence constitutively, or differentially with respect tothe cell, tissue, organ or developmental stage at which expressionoccurs, in response to external stimuli such as physiological stresses,pathogens, or metal ions, amongst others, or in response to one or moretranscriptional activators. As such, the promoter used in accordancewith the methods of the present invention may include a constitutivepromoter, an inducible promoter, a tissue-specific promoter or anactivatable promoter.

The present invention contemplates the use of any promoter which isactive in a cell of interest. As such, a wide array of promoters whichare active in any of bacteria, fungi, animal cells or plant cells wouldbe readily ascertained by one of ordinary skill in the art. However, insome embodiments, plant cells are used. In these embodiments,plant-active constitutive, inducible, tissue-specific or activatablepromoters are typically used.

Plant constitutive promoters typically direct expression in nearly alltissues of a plant and are largely independent of environmental anddevelopmental factors. Examples of constitutive promoters that may beused in accordance with the present invention include plant viralderived promoters such as the Cauliflower Mosaic Virus 35S and 19S (CaMV35S and CaMV 19S) promoters; bacterial plant pathogen derived promoterssuch as opine promoters derived from Agrobacterium spp., eg. theAgrobacterium-derived nopaline synthase (nos) promoter; andplant-derived promoters such as the rubisco small subunit gene (rbcS)promoter, the plant ubiquitin promoter (Pubi), the rice actin promoter(Pact) and the oat globulin promoter.

“Inducible” promoters include, but are not limited to, chemicallyinducible promoters and physically inducible promoters. Chemicallyinducible promoters include promoters which have activity that isregulated by chemical compounds such as alcohols, antibiotics, steroids,metal ions or other compounds. Examples of chemically induciblepromoters include: alcohol regulated promoters (eg. see European Patent637 339); tetracycline regulated promoters (eg. see U.S. Pat. No.5,851,796 and U.S. Pat. No. 5,464,758); steroid responsive promoterssuch as glucocorticoid receptor promoters (eg. see U.S. Pat. No.5,512,483), estrogen receptor promoters (eg. see European PatentApplication 1 232 273), ecdysone receptor promoters (eg. see U.S. Pat.No. 6,379,945) and the like; metal-responsive promoters such asmetallothionein promoters (eg. see U.S. Pat. No. 4,940,661, U.S. Pat.No. 4,579,821 and U.S. Pat. No. 4,601,978); and pathogenesis relatedpromoters such as chitinase or lysozyme promoters (eg. see U.S. Pat. No.5,654,414) or PR protein promoters (eg. see U.S. Pat. No. 5,689,044,U.S. Pat. No. 5,789,214, Australian Patent 708850, U.S. Pat. No.6,429,362).

The inducible promoter may also be a physically regulated promoter whichis regulated by non-chemical environmental factors such as temperature(both heat and cold), light and the like. Examples of physicallyregulated promoters include heat shock promoters (eg. see U.S. Pat. No.5,447,858, Australian Patent 732872, Canadian Patent Application1324097); cold inducible promoters (eg. see U.S. Pat. No. 6,479,260,U.S. Pat. No. 6,084,08, U.S. Pat. No. 6,184,443 and U.S. Pat. No.5,847,102); light inducible promoters (eg. see U.S. Pat. No. 5,750,385and Canadian Patent 132 1563); light repressible promoters (eg. see NewZealand Patent 508103 and U.S. Pat. No. 5,639,952).

“Tissue specific promoters” include promoters which are preferentiallyor specifically expressed in one or more specific cells, tissues ororgans in an organism and/or one or more developmental stages of theorganism. It should be understood that a tissue specific promoter may,in some cases, also be inducible.

Examples of plant tissue specific promoters include: root specificpromoters such as those described in US Patent Application 2001047525;fruit specific promoters including ovary specific and receptacle tissuespecific promoters such as those described in European Patent 316 441,U.S. Pat. No. 5,753,475 and European Patent Application 973 922; andseed specific promoters such as those described in Australian Patent612326 and European Patent application 0 781 849 and Australian Patent746032.

In some embodiments, the tissue specific promoter is a seed and/or grainspecific promoter. Exemplary seed or grain specific promoters includepuroindoline-b gene promoters (for example see Digeon et al., Plant Mol.Biol. 39: 1101-1112, 1999); Pbf gene promoters (for example see Mena etal., Plant J. 16: 53-62, 1998); GS₁₋₂ gene promoters (for example seeMuhitch et al., Plant Sci. 163: 865-872, 2002); glutelin or Gt1 genepromoters (for example see Okita et al., J. Biol. Chem. 264:12573-12581, 1989; Zheng et al., Plant J. 4: 357-366, 1993; Sindhu etal., Plant Sci. 130: 189-196, 1997; Nandi et al., Plant Sci. 163:713-722, 2002); Hor2-4 gene promoters (for example see Knudsen andMüller, Planta 195: 330-336, 1991; Patel et al., Mol. Breeding 6:113-123, 2000; Wong et al., Proc. Natl. Acad. Sci. USA 99: 16325-16330,2002); lipoxygenase 1 gene promoters (for example see Rouster et al.,Plant J. 15: 435-440, 1998); Chi26 gene promoters (for example see Leahet al., Plant J. 6: 579-589, 1994); Glu-D1-1 gene promoters (for examplesee Lamacchia et al., J. Exp. Bot. 52: 243-250, 2001; Zhang et al.,Theor. Appl. Genet. 106: 1139-1146, 2003); Hor3-1 gene promoters (forexample see Sörensen et al., Mol. Gen. Genet. 250: 750-760, 1996;Horvath et al., Proc. Natl. Acad. Sci. USA 97: 1914-1919, 2000) and Waxy(Wx) gene promoters (for example see Yao et al., Acta Phytophysiol. Sin.22: 431-436, 1996; Terada et al., Plant Cell Physiol. 41: 881-888, 2000;Liu et al., Transgenic Res. 12: 71-82, 2003). In one specificembodiment, the seed specific promoter is an endosperm specificpromoter.

The promoter may also be a promoter that is activatable by one or moredefined transcriptional activators, referred to herein as an“activatable promoter”. For example, the activatable promoter maycomprise a minimal promoter operably connected to an Upstream ActivatingSequence (UAS), which comprises, inter alia, a DNA binding site for oneor more transcriptional activators.

As referred to herein the term “minimal promoter” should be understoodto include any promoter that incorporates at least an RNA polymerasebinding site and, preferably a TATA box and transcription initiationsite and/or one or more CAAT boxes. When the cell is a plant cell, theminimal promoter may be derived from, for example, the CauliflowerMosaic Virus 35S (CaMV 35S) promoter. The CaMV 35S derived minimalpromoter may comprise, for example, a sequence that corresponds topositions −90 to +1 (the transcription initiation site) of the CaMV 35Spromoter (also referred to as a −90 CaMV 35S minimal promoter), −60 to+1 of the CaMV 35S promoter (also referred to as a −60 CaMV 35S minimalpromoter) or −45 to +1 of the CaMV 35S promoter (also referred to as a−45 CaMV 35S minimal promoter).

As set out above, the activatable promoter may comprise a minimalpromoter fused to an Upstream Activating Sequence (UAS). The UAS may beany sequence that can bind a transcriptional activator to activate theminimal promoter. Exemplary transcriptional activators include, forexample: yeast derived transcription activators such as Gal4, Pdr1, Gcn4and Ace1; the viral derived transcription activator, VP16; Hap1 (Hach etal., J Biol Chem 278: 248-254, 2000); Gaf1 (Hoe et al., Gene 215(2):319-328, 1998); E2F (Albani et al., J Biol Chem 275: 19258-19267, 2000);HAND2 (Dai and Cserjesi, J Biol Chem 277: 12604-12612, 2002); NRF-1 andEWG (Herzig et al., J Cell Sci 113: 4263-4273, 2000); P/CAF (Itoh etal., Nucl Acids Res 28: 4291-4298, 2000); MafA (Kataoka et al., J BiolChem 277: 49903-49910, 2002); human activating transcription factor 4(Liang and Hai, J Biol Chem 272: 24088-24095, 1997); Bcl10 (Liu et al.,Biochem Biophys Res Comm 320(1): 1-6, 2004); CREB-H (Omori et al., NuclAcids Res 29: 2154-2162, 2001); ARR1 and ARR2 (Sakai et al., Plant J24(6): 703-711, 2000); Fos (Szuts and Bienz, Proc Natl Acad Sci USA 97:5351-5356, 2000); HSF4 (Tanabe et al., J Biol Chem 274: 27845-27856,1999); MAML1 (Wu et al., Nat Genet 26: 484-489, 2000).

In some embodiments, the UAS comprises a nucleotide sequence that isable to bind to at least the DNA-binding domain of the GAL4transcriptional activator. UAS sequences, which can bind transcriptionalactivators that comprise at least the GAL4 DNA binding domain, arereferred to herein as UASc. In a particular embodiment, the UASccomprises the sequence 5′-CGGAGTACTGTCCTCCGAG-3′ or a functional homologthereof.

As referred to herein, a “functional homolog” of the UASc sequenceshould be understood to refer to any nucleotide sequence which can bindat least the GAL4 DNA binding domain and which may comprise a nucleotidesequence having at least 50% identity, at least 65% identity, at least80% identity or at least 90% identity with the UASc nucleotide sequence.

The UAS sequence in the activatable promoter may comprise a plurality oftandem repeats of a DNA binding domain target sequence. For example, inits native state, UASc comprises four tandem repeats of the DNA bindingdomain target sequence. As such, the term “plurality” as used hereinwith regard to the number of tandem repeats of a DNA binding domaintarget sequence should be understood to include at least 2, at least 3or at least 4 tandem repeats.

As mentioned above, the control sequences may also include a terminator.The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences generally containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. As with promotersequences, the terminator may be any terminator sequence which isoperable in the cells, tissues or organs in which it is intended to beused. Examples of suitable terminator sequences which may be useful inplant cells include: the nopaline synthase (nos) terminator, the CaMV35S terminator, the octopine synthase (ocs) terminator, potatoproteinase inhibitor gene (pin) terminators, such as the pinII andpinIII terminators and the like.

Modulating the level of (1,3;1,4)-β-D-glucan in a cell, by modulatingthe level and/or activity of a CslH-encoded (1,3;1,4)-β-D-glucansynthase in the cell, has several industrial applications.

For example, (1,3;1,4)-β-D-glucans are known to form viscous solutions.The viscosity-generating properties of soluble cereal(1,3;1,4)-β-D-glucans are critical determinants in many aspects ofcereal processing. For example, incompletely degraded(1,3;1,4)-β-D-glucans from malted barley and cereal adjuncts cancontribute to wort and beer viscosity and are associated with problemsin wort separation and beer filtration (eg. see Bamforth, Brew. Dig. 69(5): 12-16, 1994) Therefore, for example, in some embodiments, thepresent invention may be applied to reduce the level of(1,3;1,4)-β-D-glucan in barley grain, by reducing the level and/oractivity of a CslH-encoded (1,3;1,4)-β-D-glucan synthase in one or morecells of the barley grain, to increase its suitability for beerproduction.

Soluble cereal (1,3;1,4)-β-D-glucans are also considered to haveantinutritive effects in monogastric animals such as pigs and poultry.The “antinutritive” effects have been attributed to the increasedviscosity of gut contents, which slows both the diffusion of digestiveenzymes and the absorption of degradative products of enzyme action.This, in turn, leads to slower growth rates. Moreover, in dietaryformulations for poultry, high (1,3;1,4)-β-D-glucan concentrations areassociated with ‘sticky’ faeces, which are indicative of the poordigestibility of the (1,3;1,4)-β-D-glucans and which may present majorhandling and hygiene problems for producers. Therefore, in anotherembodiment, the present invention may be applied to reducing the levelof (1,3;1,4)-β-D-glucan in one or more cells of a plant used for animalfeed, to improve the suitability of the plant as animal feed.

However, cereal (1,3;1,4)-β-D-glucans are important components ofdietary fibre in human and animal diets. As used herein, the term“dietary fibre” should be understood to include the edible parts ofplants or analogous carbohydrates that are resistant to digestion andabsorption in the human small intestine with complete or partialfermentation in the large intestine. “Dietary fibre” includespolysaccharides (specifically including (1,3;1,4)-β-D-glucans),oligosaccharides, lignin and associated plant substances. In at leasthuman diets, dietary fibres promote beneficial physiological effectsincluding general bowel health, laxation, blood cholesterol attenuation,and/or blood glucose attenuation.

Humans and monogastric animals produce no enzymes that degrade(1,3;1,4)-β-D-glucans, although there are indications that somedepolymerization occurs in the stomach and small intestine, presumablydue to the activity of commensal microorganisms. By comparison, thesoluble (1,3;1,4)-β-D-glucans and other non-starchy polysaccharides arereadily fermented by colonic micro-organisms and make a smallcontribution to digestible energy. In contrast to their antinutritiveeffects in monogastric animals, oat and barley (1,3;1,4)-β-D-glucans athigh concentrations in humans have beneficial effects, especially fornon-insulin-dependent diabetics, by flattening glucose and insulinresponses that follow a meal. High concentrations of(1,3;1,4)-β-D-glucans (eg. 20% w/v) in food have also been implicated inthe reduction of serum cholesterol concentrations, by lowering theuptake of dietary cholesterol or resorption of bile acids from theintestine.

Therefore, in another embodiment, the present invention may be appliedto increasing the dietary fibre content of an edible plant or edibleplant part, by increasing the level of (1,3;1,4)-β-D-glucan in theplant, or part thereof. In some embodiments, the edible plant or ediblepart of a plant is a cereal crop plant or part thereof.

(1,3;1,4)-β-D-glucans, in common with a number of other polysaccharides,in particular (1→3)-β-D-glucans, are also thought to modifyimmunological responses in humans by a process that is mediated throughbinding to receptors on cells of the reticuloendothelial system(leucocytes and macrophages). In addition, they may have the capacity toactivate the proteins of the human complement pathway, a system that isinvoked as a first line of defense before circulating antibodies areproduced.

The present invention also facilitates the production of(1,3;1,4)-β-D-glucan in a recombinant expression system. For example, a(1,3;1,4)-β-D-glucan may be recombinantly produced by introducing a CslHnucleic acid under the control of a promoter, into a cell, wherein thecell subsequently expresses a CslH-encoded (1,3;1,4)-β-D-glucan synthaseand produces (1,3;1,4)-β-D-glucan.

A vast array of recombinant expression systems that may be used toexpress a CslH nucleic acid are known in the art. Exemplary recombinantexpression systems include: bacterial expression systems such as E. coliexpression systems (reviewed in Baneyx, Curr. Opin. Biotechnol. 10:411-421, 1999; eg. see also Gene expression in recombinantmicroorganisms, Smith (Ed.), Marcel Dekker, Inc. New York, 1994; andProtein Expression Technologies: Current Status and Future Trends,Baneyx (Ed.), Chapters 2 and 3, Horizon Bioscience, Norwich, UK, 2004),Bacillus spp. expression systems (eg. see Protein ExpressionTechnologies: Current Status and Future Trends, supra, chapter 4) andStreptomyces spp. expression systems (eg. see Practical StreptomycesGenetics, Kieser et al., (Eds.), Chapter 17, John Innes Foundation,Norwich, UK, 2000); fungal expression systems including yeast expressionsystems such as Saccharomyces spp., Schizosaccharomyces pombe, Hansenulapolymorpha and Pichia spp. expression systems and filamentous fungiexpression systems (eg. see Protein Expression Technologies: CurrentStatus and Future Trends, supra, chapters 5, 6 and 7; Buckholz andGleeson, Bio/Technology 9(11): 1067-1072, 1991; Cregg et al., Mol.Biotechnol. 16(1): 23-52, 2000; Cereghino and Cregg, FEMS MicrobiologyReviews 24: 45-66, 2000; Cregg et al., Bio/Technology 11: 905-910,1993); mammalian cell expression systems including Chinese Hamster Ovary(CHO) cell based expression systems (eg. see Protein ExpressionTechnologies: Current Status and Future Trends, supra, chapter 9);insect cell cultures including baculovirus expression systems (eg. seeProtein Expression Technologies: Current Status and Future Trends,supra, chapter 8; Kost and Condreay, Curr. Opin. Biotechnol. 10:428-433, 1999; Baculovirus Expression Vectors: A Laboratory Manual WHFreeman & Co., New York, 1992; and The Baculovirus Expression System: ALaboratory Manual, Chapman & Hall, London, 1992); Plant cell expressionsystems such as tobacco, soybean, rice and tomato cell expressionsystems (eg. see review of Hellwig et al., Nat Biotechnol 22: 1415-1422,2004); and the like.

Therefore, in a third aspect, the present invention provides a methodfor producing (1,3;1,4)-β-D-glucan, the method comprising transforming acell with an isolated CslH nucleic acid and allowing the cell to expressthe isolated CslH nucleic acid.

In some embodiments, the cell is a cell from a recombinant expressionsystem as hereinbefore defined.

In a fourth aspect, the present invention also provides(1,3;1,4)-β-D-glucan produced according to the method of the thirdaspect of the invention.

In a fifth aspect, the present invention also provides a cellcomprising:

-   -   a modulated level and/or activity of CslH-encoded        (1,3;1,4)-β-D-glucan synthase relative to a wild type cell of        the same taxon; and/or    -   modulated expression of a CslH nucleic acid relative to a wild        type cell of the same taxon.

In some embodiments, the cell further comprises a modulated level of(1,3;1,4)-β-D-glucan relative to a wild type cell of the same taxon.

In some embodiments, the cell of the fifth aspect of the invention isproduced according to the methods of the first or second aspects of thepresent invention as described herein. In further embodiments, the cellis a plant cell, a monocot plant cell, a Poales plant cell and/or acereal crop plant cell.

Furthermore, in a sixth aspect, the present invention provides amulticellular structure comprising one or more cells according to thefifth aspect of the invention.

As referred to herein, a “multicellular structure” includes anyaggregation of one or more cells. As such, the term “multicellularstructure” specifically encompasses tissues, organs, whole organisms andparts thereof. Furthermore, a multicellular structure should also beunderstood to encompass multicellular aggregations of cultured cellssuch as colonies, plant calli, suspension cultures and the like.

As mentioned above, in some embodiments of the invention, the cell is aplant cell and as such, the present invention includes a whole plant,plant tissue, plant organ, plant part, plant reproductive material orcultured plant tissue, comprising one or more plant cells according tothe sixth aspect of the invention.

In another embodiment, the present invention provides a cereal cropplant comprising one or more cells according to the fifth aspect of theinvention.

In a particular embodiment, the present invention provides cereal graincomprising one or more cells according to the fifth aspect of theinvention.

Therefore, in a seventh aspect, the present invention provides a cerealgrain comprising a modulated level of (1,3;1,4)-β-D-glucan, wherein thegrain comprises one or more cells comprising a modulated level and/oractivity of a CslH-encoded (1,3;1,4)-β-D-glucan synthase and/ormodulated expression of a CslH nucleic acid.

In some embodiments, the grain may have an increased level of(1,3;1,4)-β-D-glucan compared to wild type grain from the same species.In alternate embodiments, the grain may have a decreased level of(1,3;1,4)-β-D-glucan compared to wild type grain from the same species.

In some embodiments wherein the grain is a wheat grain, the wheat graincomprises level of (1,3;1,4)-β-D-glucan of at least 1%, at least 1.1%,at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least1.6%, at least 1.7%, at least 1.8% or 1.9% on a fresh weight basis ofair dried whole grain.

In an eighth aspect, the present invention also provides flourcomprising:

-   -   flour produced by the milling of the grain of the seventh aspect        of the invention; and    -   optionally, flour produced by the milling of one or more other        grains.

As such, the flour produced by the milling of the grain of the seventhaspect of the invention may comprise, for example approximately 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% by weight of the flour ofthe ninth aspect of the invention.

As referred to herein “milling” contemplates any method known in the artfor milling grain, such as those described by Brennan et al. (Manual ofFlour and Husk Milling, Brennan et al. (Eds.), AgriMedia, ISBN:3-86037-277-7).

In some embodiments, the flour produced by the milling of the grain ofthe seventh aspect of the invention used in the flour comprises anincreased level of (1,3;1,4)-β-D-glucan compared to wild type flour.

The “flour produced by the milling of one or more other grains” may beflour produced by milling grain derived from any cereal plant, ashereinbefore defined. This component of the flour of the eighth aspectof the invention may, for example, comprise 0%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80% or 90% by weight.

In some embodiments, the flour produced by the milling of one or moreother grains is wheat flour and, therefore, the flour of the eighthaspect of the invention may be particularly suitable for producingbread, cakes, biscuits and the like.

As set out above, the present invention is predicated, in part, on theidentification and isolation of CslH nucleotide sequences and CslH aminoacid sequences that encode (1,3;1,4)-β-D-glucan synthases.

Therefore, in a ninth aspect, the present invention provides an isolatedCslH nucleic acid as hereinbefore defined, or a complement, reversecomplement or fragment thereof.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be isolated because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.An “isolated” nucleic acid molecule should also be understood to includea synthetic nucleic acid molecule, including those produced by chemicalsynthesis using known methods in the art or by in-vitro amplification(eg. polymerase chain reaction and the like).

The isolated nucleic acid molecules of the present invention maycomprise any polyribonucleotide or polydeoxyribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example, the isolatednucleic acid molecules of the invention may comprise single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and/or double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the isolated nucleic acid molecules may comprise oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Theisolated nucleic acid molecules may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

As set out above, the present invention also provides fragments of anucleotide sequence. “Fragments” of a nucleotide sequence should be atleast 15, 20, 30, 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400,450, 500, 550, or 600 nucleotides (nt) in length. These fragments havenumerous uses that include, but are not limited to, diagnostic probesand primers. Of course, larger fragments, such as those of 601-3000 ntin length are also useful according to the present invention as arefragments corresponding to most, if not all, of a CslH nucleic acid.

In some embodiments, the fragment may comprise a functional fragment ofa CslH nucleic acid. That is, the polynucleotide fragments of theinvention may encode a polypeptide having (1,3;1,4)-β-D-glucan synthasefunctional activity as defined herein.

In a tenth aspect, the present invention provides a genetic construct orvector comprising an isolated nucleic acid molecule of the ninth aspectof the invention.

The vector or construct may further comprise one or more of: an originof replication for one or more hosts; a selectable marker gene which isactive in one or more hosts; or one or more control sequences whichenable transcription of the isolated nucleic acid molecule in a cell.

“Selectable marker genes” include any nucleotide sequences which, whenexpressed by a cell, confer a phenotype on the cell that facilitates theidentification and/or selection of these transformed cells. A range ofnucleotide sequences encoding suitable selectable markers are known inthe art. Exemplary nucleotide sequences that encode selectable markersinclude: antibiotic resistance genes such as ampicillin-resistancegenes, tetracycline-resistance genes, kanamycin-resistance genes, theAURI-C gene which confers resistance to the antibiotic aureobasidin A,neomycin phosphotransferase genes (eg. nptI and nptII) and hygromycinphosphotransferase genes (eg. hpt); herbicide resistance genes includingglufosinate, phosphinothricin or bialaphos resistance genes such asphosphinothricin acetyl transferase encoding genes (eg. bar), glyphosateresistance genes including 3-enoyl pyruvyl shikimate 5-phosphatesynthase encoding genes (eg. aroA), bromyxnil resistance genes includingbromyxnil nitrilase encoding genes, sulfonamide resistance genesincluding dihydropterate synthase encoding genes (eg. sul) andsulfonylurea resistance genes including acetolactate synthase encodinggenes; enzyme-encoding reporter genes such as GUS andchloramphenicolacetyltransferase (CAT) encoding genes; fluorescentreporter genes such as the green fluorescent protein-encoding gene; andluminescence-based reporter genes such as the luciferase gene, amongstothers.

Furthermore, it should be noted that the selectable marker gene may be adistinct open reading frame in the construct or may be expressed as afusion protein with the CslH-encoded (1,3;1,4)-β-D-glucan synthasepolypeptide.

The tenth aspect of the invention extends to all genetic constructsessentially as described herein, which include further nucleotidesequences intended for the maintenance and/or replication of the geneticconstruct in prokaryotes or eukaryotes and/or the integration of thegenetic construct or a part thereof into the genome of a eukaryotic orprokaryotic cell.

In some embodiments, the vector or construct is adapted to be at leastpartially transferred into a plant cell via Agrobacterium-mediatedtransformation. Accordingly, the vector or construct may comprise leftand/or right T-DNA border sequences.

Suitable T-DNA border sequences would be readily ascertained by one ofskill in the art. However, the term “T-DNA border sequences” may includesubstantially homologous and substantially directly repeated nucleotidesequences that delimit a nucleic acid molecule that is transferred froman Agrobacterium sp. cell into a plant cell susceptible toAgrobacterium-mediated transformation. By way of example, reference ismade to the paper of Peralta and Ream (Proc. Natl. Acad. Sci. USA,82(15): 5112-5116, 1985) and the review of Gelvin (Microbiology andMolecular Biology Reviews, 67(1): 16-37, 2003).

Although in some embodiments, the vector or construct is adapted to betransferred into a plant via Agrobacterium-mediated transformation, thepresent invention also contemplates any suitable modifications to thegenetic construct which facilitate bacterial mediated insertion into aplant cell via bacteria other than Agrobacterium sp., for example, asdescribed in Broothaerts et al. (Nature 433: 629-633, 2005).

Those skilled in the art will be aware of how to produce the constructsdescribed herein and of the requirements for obtaining the expressionthereof, when so desired, in a specific cell or cell-type under theconditions desired. In particular, it will be known to those skilled inthe art that the genetic manipulations required to perform the presentinvention may require the propagation of a genetic construct describedherein or a derivative thereof in a prokaryotic cell such as an E. colicell or a plant cell or an animal cell. Exemplary methods for cloningnucleic acid molecules are described in Sambrook et al. (2000, supra)

In an eleventh aspect, the present invention provides a cell comprisingthe isolated nucleic acid molecule of the ninth aspect of the inventionor genetic construct of the tenth aspect of the invention.

The isolated nucleic acid molecule of the tenth or eleventh aspects ofthe invention or genetic construct of the twelfth aspect of theinvention may be introduced into a cell via any means known in the art.

The isolated nucleic acid molecule or construct referred to above may bemaintained in the cell as a DNA molecule, as part of an episome (eg. aplasmid, cosmid, artificial chromosome or the like) or it may beintegrated into the genomic DNA of the cell.

As used herein, the term “genomic DNA” should be understood in itsbroadest context to include any and all DNA that makes up the geneticcomplement of a cell. As such, the genomic DNA of a cell should beunderstood to include chromosomes, mitochondrial DNA, plastid DNA,chloroplast DNA, endogenous plasmid DNA and the like. As such, the term“genomically integrated” contemplates chromosomal integration,mitochondrial DNA integration, plastid DNA integration, chloroplast DNAintegration, endogenous plasmid integration, and the like.

The isolated nucleic acid molecule may be operably connected to, interalia, a control sequence and/or a promoter such that the cell mayexpress the isolated nucleic acid molecule.

The cell may be any prokaryotic or eukaryotic cell. As such, the cellmay be a prokaryotic cell such as a bacterial cell including an E. colicell or an Agrobacterium spp. cell, or an archaea cell. The cell mayalso be a eukaryotic cell including a fungal cell such as a yeast cellor mycelial fungus cell; an animal cell such as a mammalian cell or aninsect cell; or a plant cell. In a particular embodiment, the cell is aplant cell. In some embodiments, the plant cell is a monocot plant cell,a Poales plant cell, or a cereal crop plant cell.

In a twelfth aspect, the present invention provides a multicellularstructure, as hereinbefore defined, comprising one or more of the cellsof the eleventh aspect of the invention.

As mentioned above, in some embodiments, the cell is a plant cell and assuch, the present invention should be understood to specifically includea whole plant, plant tissue, plant organ, plant part, plant reproductivematerial, or cultured plant tissue, comprising one or more cells of theeleventh aspect of the invention.

In a further embodiment, the present invention provides a monocot plant,a Poales plant or a cereal crop plant or part thereof, comprising one ormore cells of the eleventh aspect of the invention.

In some embodiments, the present invention provides cereal graincomprising one or more cells of the eleventh aspect of the invention.

As set out above, the present invention also provides amino acidsequences for CslH-encoded (1,3;1,4)-β-D-glucan synthases. Accordingly,in a thirteenth aspect, the present invention provides an isolatedCslH-encoded (1,3;1,4)-β-D-glucan synthase as hereinbefore defined, or afragment thereof.

The isolated polypeptides may comprise of amino acids joined to eachother by peptide bonds or modified peptide bonds, ie., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. The isolated polypeptides of the present invention may bemodified by either natural processes, such as post-translationalprocessing, or by chemical modification techniques which are well knownin the art.

Modifications can occur anywhere in the isolated polypeptide, includingthe peptide backbone, the amino acid side-chains and/or the termini. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given isolatedpolypeptide. Also, an isolated polypeptide of the present invention maycontain many types of modifications.

The polypeptides may be branched, for example, as a result ofubiquitination, and/or they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result frompost-translation natural processes or may be made by synthetic methods.

Modifications include acetylation, acylation, ADP-ribosylation,amidation, biotinylation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphatidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,PEGylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, Proteins—Structure And Molecular Properties 2^(nd)Ed., Creighton (ed.), W. H. Freeman and Company, New York, 1993);Posttranslational Covalent Modification Of Proteins, Johnson (Ed.),Academic Press, New York, 1983; Seifter et al., Meth Enzymol 182:626-646, 1990); Rattan et al., Ann NY Acad Sci 663: 48-62, 1992).

As set out above, the present invention also provides fragments ofisolated polypeptides. Polypeptide fragments may be “free-standing” orcomprised within a larger polypeptide of which the fragment forms a partor region.

The polypeptide fragments can be at least 3, 4, 5, 6, 8, 9, 10, 11, 12,13, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, or 150 amino acids in length.

In some embodiments, the fragment is a functional fragment and thuscomprises (1,3;1,4)-β-D-glucan synthase functional activity. However,even if the fragment does not retain one or more biological functions ofa CslH-encoded (1,3;1,4)-β-D-glucan synthase, other functionalactivities may still be retained. For example, the fragments may lackCslH-encoded (1,3;1,4)-β-D-glucan synthase functional activity butretain the ability to induce and/or bind to antibodies which recognizethe complete or mature forms of an isolated CslH-encoded(1,3;1,4)-β-D-glucan synthase polypeptide. A peptide, polypeptide orprotein fragment which has the ability to induce and/or bind toantibodies which recognize the complete or mature forms of the isolatedCslH-encoded (1,3;1,4)-β-D-glucan synthase polypeptide is referred toherein as a “CslH-encoded (1,3;1,4)-β-D-glucan synthase epitope”.

A CslH-encoded (1,3;1,4)-β-D-glucan synthase epitope may comprise as fewas three or four amino acid residues. In some embodiments the epitopemay comprise, for example, at least 5, at least 10, at least 20, atleast 50, at least 100 or at least 200 amino acid residues. Whether aparticular epitope of an isolated CslH-encoded (1,3;1,4)-β-D-glucansynthase polypeptide retains such immunologic activities can readily bedetermined by methods known in the art. As such, one CslH-encoded(1,3;1,4)-β-D-glucan synthase polypeptide fragment is a polypeptidecomprising one or more CslH-encoded (1,3;1,4)-β-D-glucan synthaseepitopes.

A polypeptide comprising one or more CslH-encoded (1,3;1,4)-β-D-glucansynthase epitopes may be produced by any conventional means for makingpolypeptides including synthetic and recombinant methods known in theart. In some embodiments, CslH-encoded (1,3;1,4)-β-D-glucan synthaseepitope-bearing polypeptides may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor the synthesis of large numbers of peptides (Houghten, Proc. Natl.Acad. Sci. USA 82: 5131-5135, 1985).

The isolated CslH-encoded (1,3;1,4)-β-D-glucan synthase polypeptides andCslH-encoded (1,3;1,4)-β-D-glucan synthase epitope-bearing polypeptidesare useful, for example, in the generation of antibodies that bind tothe isolated CslH-encoded (1,3;1,4)-β-D-glucan synthase polypeptides ofthe invention.

Such antibodies are useful, inter alia, in the detection andlocalization of (1,3;1,4)-β-D-glucan synthase polypeptides and inaffinity purification of (1,3;1,4)-β-D-glucan synthase polypeptides. Theantibodies may also routinely be used in a variety of qualitative orquantitative immunoassays using methods known in the art. For examplesee Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press 2^(nd) Ed., 1988).

Accordingly, in a fourteenth aspect, the present invention provides anantibody or an epitope binding fragment thereof, raised against anisolated CslH-encoded (1,3;1,4)-β-D-glucan synthase polypeptide ashereinbefore defined or an epitope thereof.

The antibodies of the present invention include, but are not limited to,polyclonal, monoclonal, multispecific, chimeric antibodies, single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library and epitope-binding fragments of any of the above.

The term “antibody”, as used herein, refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen-binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

The antibodies of the present invention may be monospecific, bispecific,trispecific, or of greater multispecificity. Multispecific antibodiesmay be specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. For example, see PCT publicationsWO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J.Immunol. 147: 60-69, 1991; U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; and Kostelny et al. J. Immunol. 148:1547-1553, 1992).

In some embodiments, the antibodies of the present invention may act asagonists or antagonists of CslH-encoded (1,3;1,4)-β-D-glucan synthase.In further embodiments, the antibodies of the present invention may beused, for example, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofCslH-encoded (1,3;1,4)-β-D-glucan synthase in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory Press, 2nd ed. 1988).

The term “antibody”, as used herein, should be understood to encompassderivatives that are modified, eg. by the covalent attachment of anytype of molecule to the antibody such that covalent attachment does notprevent the antibody from binding to a CslH-encoded (1,3;1,4)-β-D-glucansynthase or an epitope thereof. For example, the antibody derivativesinclude antibodies that have been modified, eg., by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. Furthermore, any of numerouschemical modifications may also be made using known techniques. Theseinclude specific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

Antibodies may be generated using methods known in the art.

For example, if in vivo immunization is used, animals may be immunizedwith free peptide; however, anti-peptide antibody titer may be boostedby coupling of the peptide to a macromolecular carrier, such as keyholelimpet hemacyanin (KLH) or tetanus toxoid. For example, peptidescontaining cysteine residues may be coupled to a carrier using a linkersuch as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carriers using a more general linking agentsuch as glutaraldehyde.

Animals such as rabbits, rats and mice may be immunized with either freeor carrier-coupled peptides, for instance, by intraperitoneal and/orintradermal injection of emulsions containing about 100 micrograms ofpeptide or carrier protein and Freund's adjuvant. Several boosterinjections may be needed, for example, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Polyclonal antibodies to a CslH-encoded (1,3;1,4)-β-D-glucan synthasepolypeptide or a polypeptide comprising one or more CslH-encoded(1,3;1,4)-β-D-glucan synthase epitopes can be produced by variousprocedures well known in the art. For example, a polypeptide of theinvention can be administered to various host animals including, but notlimited to, rabbits, mice, rats, etc. to induce the production of seracontaining polyclonal antibodies specific for the antigen. Variousadjuvants may be used to increase the immunological response, dependingon the host species, for example, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Such adjuvants are also well known in the art.

As another example, monoclonal antibodies can be prepared using a widevariety of techniques known in the art including the use of hybridoma,recombinant, and phage display technologies, or a combination thereof.For example, monoclonal antibodies can be produced using hybridomatechniques including those known in the art and taught, for example, inHarlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nd ed., 1988) and Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas (Elsevier, NY, 1981). The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. For example,mice can be immunized with a polypeptide of the invention or a cellexpressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well-known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Antibody fragments which recognize one or more CslH-encoded(1,3;1,4)-β-D-glucan synthase epitopes may also be generated by knowntechniques. For example, Fab and F(ab′)2 fragments may be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments). F(ab′)2 fragments contain the variable region, the lightchain constant region and the CH1 domain of the heavy chain.

The antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In a particular embodiment, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labelled antigen or antigen boundor captured to a solid surface or bead. Phages used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein.

Examples of phage display methods include those disclosed by Brinkman etal. (J. Immunol. Methods 182: 41-50, 1995), Ames et al. (J. Immunol.Methods 184: 177-186, 1995), Kettleborough et al. (Eur. J. Immunol. 24:952-958, 1994), Persic et al. (Gene 187: 9-18, 1997), Burton et al.(Advances in Immunology 57: 191-280, 1994); PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

After phage selection, the antibody coding regions from the phage can beisolated and used to generate whole antibodies or any other desiredantigen binding fragment, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria. Forexample, techniques to recombinantly produce Fab, Fab′ and F(ab′)2fragments can also be employed using methods known in the art such asthose disclosed in PCT publication WO 92/22324; Mullinax et al.(BioTechniques 12(6): 864-869, 1992); and Sawai et al. (AJRI 34:26-34,1995); and Better et al. (Science 240: 1041-1043, 1988).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (Methods in Enzymology 203: 46-88, 1991); Shuet al. (Proc. Natl. Acad. Sci. USA 90: 7995-7999, 1993); and Skerra etal. (Science 240: 1038-1040, 1988).

The present invention is further described by the following non-limitingexamples:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (A) a schematic of the T-DNA of the HvCslH1::pGBW15construct used in gain-of-function experiments in Arabidopsis. AfterGateway cloning, the 3×HA tag was attached at the NH₂-terminal end ofthe full-length HvCslH1 ORF. (B) Transcript levels in the leaves ofmature HvCslH1 transgenic T1 plants as determined by Northern blotanalysis. Upper panel, X-ray film exposure; lower panel, correspondingethidium bromide-stained gel. The observed 2.5 kb transcript sizecorresponds to the expected size of the tagged HvCslH1 mRNA. (C)3×HA-tagged HvCslH1 protein levels in 3-week-old pooled HvCslH1transgenic T2 lines as determined by Western blot analysis. Thirtymicrograms of mixed microsomal membrane protein was loaded per lane andblots probed with the anti-HA antibody. B and C; Numbers refer totransgenic lines, Col-0, wild-type untransformed line. Col-0, lines 8and 14 from the same blot, all other lines are from different blots.

FIG. 2 shows transmission electron micrographs showing detection ofβ-glucan in walls of HvCslH1-expressing lines with a β-glucan-specificmonoclonal antibody (Meikle et al., Plant J 5: 1-9, 1994). (A-C) line 8,16, 11; (D) wild-type Col-0 control; (E) line 6. A and D show cells ofthe vascular bundle; B and C, mesophyll cells; E, epidermal cells. Scalebar=0.5 μm (A-C, E), 1 (D).

FIG. 3 shows HPAEC profiles of oligosaccharides released upon(1,3;1,4)-β-D-glucan endo-hydrolase digestion of alcohol insolubleresidue (AIR) prepared from 145 day-old Arabidopsis line 16-1 rosetteleaf tissue (

). 16-1 pre-enzyme treatment buffer wash (

). Barley mature leaf (entire sheath) AIR was used as the positivecontrol sample (

) G4G3G_(R) (3-O-(3-cellobiosyl D-glucose, DP3) and G4G4G3G_(R)(3-O-β-cellotriosyl D-glucose, DP4) peaks are indicated.

FIG. 4 shows transmission electron micrographs showing the detection ofthe 3×HA-tagged HvCslH1 protein by a gold-labelled anti-HA antibody insections of high pressure-frozen leaves of Arabidopsis transgenic line11. (A and B) mesophyll cells. G, Golgi body, cw, cell wall, v, vacuole,er, endoplasmic reticulum. Scale bar=0.5 μm (A), 0.2 μm (B). Arrowsindicate Golgi-associated vesicle labeling.

FIG. 5 shows HvCslH1 expression in barley as determined by QPCR and insitu PCR analyses. (A) Normalised levels of HvCslH1 transcript(copies/microlitre cDNA) in a range of barley tissues. Control genes fornormalisation were GAPDH, cyclophilin and α-tubulin. (B) Normalisedlevels of HvCslH1 transcript in developing endosperm 0-24 dayspost-pollination. Control genes were GAPDH, α-tubulin and elongationfactor-1a. (C) Normalised levels of HvCslH1 transcript in 10 day-oldfirst leaf. Control genes were GAPDH, cyclophilin and HSP70. Error barson QPCR plots indicate standard deviation. (D-F) In situ PCR images ofthe maturing zone of a 7 day-old first leaf using probes for 18S RNA(positive control, D), HvCslH1 (F) and a negative control (E). Scalebar=100 μm.

FIG. 6 shows structural features of HvCslH1. (A) Exon-intron structureof HvCslH1. Black bars indicate exons, the thin black line introns and5′ and 3′ UTRs. Numbers above boxes show size of exons, numbers belowthe line show intron size. Italicised numbers refer to the size of 5′and 3′ UTRs, bold-underline, the length of known sequence upstream ofthe start codon. Numbers are in base pairs. Thick black bars indicatethe six consensus trans-membrane domains as predicted by ARAMEMNON(http://aramemnon.botanik.uni-koeln.de/). (B) Kyte-Doolittlehydrophobicity plot (Kyte and Doolittle, J Mol Biol 157: 105-132, 1982)of HvCslH1. A 19 amino acid window with a +1.6 cutoff was used. The sixpredicted transmembrane domains are indicated by black bars. Numbersrefer to amino acids. (C) Predicted membrane topology of HvCslH1. NH₂,amino terminal; COOH, carboxy terminal; lumen, interior of ER, Golgibody or vesicle; cyt, cytoplasm, mem, membrane, D,D,D,QXXRW, signaturemotif of CAZy GT2 family. Sequence of the QXXRW motif in HvCslH1 isQFKRW.

FIG. 7 shows a phylogenetic tree of full-length barley (Hordeum vulgare)and rice (Oryza sativa) CSLH sequences. A. thaliana and poplar (Populustrichocarpa) CSLB protein sequences are included because the CSLB familyis the most closely related of the CSL families to the CSLH family. Thealignment was generated using ClustalX and the in-built distancealgorithm with neighbour joining used. The number of bootstrapreplicates (from a total of 1,000) supporting each Glade is indicatedbelow the internode for that Glade. Accession numbers are: HvCslH1(FJ459581), OsCSLH1 (Os10g20090, AC119148), OsCSLH2 (Os04g35020,AL606632), OsCSLH3 (Os04g35030, AL606632), PtCSLB1(http://genome.jgi-psf.org/Poptr1 1/Poptr1 1.home.html; ID no. 572982),PtCSLB2 (ID no. 684214), AtCSLB1 (At2G32610, NM_(—)128820), AtCSLB2(At2G32620, NM_(—)128821), AtCSLB3 (At2G32530, NM_(—)179859), AtCSLB4(At2G32540, NM_(—)128813), AtCSLB5 (At4G15290, NM_(—)117617, AtCSLB6(At4G15320, NM_(—)117620).

FIG. 8 shows a partial genomic map of the short arm of chromosome 2Hwhere HvCslH1 is located. HvCslH1 and a cluster of four HvCSLF geneswere mapped to an interval corresponding to 69.2-71.5 Mb on theSteptoe×Morex bin map close to the centromere (indicated by a blackcircle). HvCslH1 was placed in bin 8, co-segregating with the wg996marker. On the Steptoe×Morex reference map, wg996 co-segregates withabc162 and is 2.3 cM south of abc468, the marker that co-segregates withthe four HvCSLF genes (Burton et al., Plant Physiol 146: 1821-1833,2008). Key markers are shown on the left, their distances from the topof the chromosome in centimorgans (cM) and the LOD (logarithm of theodds to the base 10) score in the malt β-glucan QTL analysis of Han etal. (Theor Appl Genet 91: 921-927, 1995) are indicated on the right.

FIG. 9 shows (A) HPAEC profiles of oligosaccharides released upon(1,3;1,4)-β-D-glucan endo-hydrolase digestion of AIR prepared from 145day-old combined leaf and stem material from Arabidopsis line 16-2 (

). 16-2 pre-enzyme treatment buffer wash (

). Barley mature leaf (entire sheath) AIR was used as the positivecontrol sample (

). Laminaribiose standard (

). Retention times for maltose (Gα4G) and cellobiose (Gβ4G) are alsomarked by arrows. (B) MALDI-TOF MS chromatogram of enzyme-digested AIRof sample in A. DP2 (laminaribiose), DP3 (3-O-β-cellobiosyl D-glucose)and DP4 (3-O-β-cellotriosyl D-glucose) peaks are indicated.

FIG. 10 shows the nucleotide sequence identity, protein sequenceidentity and protein sequence similarity between CslH sequences derivedfrom Barley (Hordeum vulgare) and Rice (Oryza sativa).

FIG. 11 shows a ClustalW multiple sequence alignment of CslH amino acidsequences derived from Barley (Hordeum vulgare) and Rice (Oryza sativa).

FIG. 12 is a phylogenetic tree showing the relationship of completeCslB, F and H amino acid sequences derived from Barley (Hordeumvulgare), Rice (Oryza sativa), Arabidopsis thaliana and poplar (Populustrichocarpa).

FIG. 13 shows transmission electron micrographs illustrating thedetection of (1,3;1,4)-β-D-glucan with a (1,3;1,4)-β-D-glucan-specificmonoclonal antibody in epidermal cell walls of four transgenicArabidopsis plant lines used as parents in OsCSLF2×HvCslH1 transgenicplant crosses. HvCslH1 line individuals 15-8-3 and 15-11-7 are shown inpanels A and B, respectively, and OsCSLF2 line individuals H37-5 andH17-4-4 in panels C and D, respectively.

FIG. 14 shows transmission electron micrographs illustrating thedetection of (1,3;1,4)-β-D-glucan with a (1,3;1,4)-β-D-glucan-specificmonoclonal antibody in cell walls of progeny resulting fromOsCSLF2×HvCslH1 transgenic plant crosses. An individual from a cross of15-8-3×H37-5 is shown in (A), a sib of 15-8-3×H37-5 (B), 15-8-3×H37-7(C), a sib of 15-8-3×H37-7 (D), 15-8-15×H37-16 (E and F),15-11-13×H37-11 (G) and 15-11-7×H17-4 (H). Panels A-E, G-H showepidermal cells, panel F, mesophyll cells.

FIG. 15 shows a vector map of the pGWB15 vector used to express the CslHgene in Arabidopsis.

FIG. 16 shows the DNA sequence and translated amino acid sequence of theCslH1 gene cDNA from barley cv. Himalaya. The DNA sequence is shownnumbered every ten bases and the translated amino acid sequence of thesingle large open reading frame is shown beneath in single letter form.

FIG. 17 shows a comparison of the barley CslH1 gene cDNA and genomicsequences against the genomic sequences of the three wheat CslH1 genehomeologs (TaCslH1-1, 1-2 and 1-3). The DNA sequences of the barley cDNA(top, HvCslH1 from cv. Schooner (SEQ ID NO: 1) and HvCslH1Him from cv.Himalaya, (SEQ ID NO: 69) and genomic clones (HvCslH1g from cv. Morex(SEQ ID NO: 9) and HvCslH1gHim from cv. Himalaya (SEQ ID NO: 71) werealigned with the three wheat sequences (TaCslH1-1, 1-2 and 1-3, (SEQ IDNO: 78, 80 and 81, respectively) in BioEdit using the Muscle comparisonprogramme. The alignment position is numbered above the sequences anddashes indicate gaps introduced to optimise the alignment. Nucleotidesidentical to the wheat genomic sequence (TaCslH1-1) are indicated bydots. The exon/intron boundaries are shown in bold in the wheat genomicsequence (TaCslH1-1). For reference, the ATG initiation codon of theCslH coding region starts at alignment position 98 and the stop codonTAA starts at position 3320, both are underlined.

FIG. 18 shows a comparison of the amino acid sequences of the barley cv.Himalaya and wheat CslH1 proteins. The translated amino acid sequencesof the barley gene (top, HvCslH1(Him) were aligned with the three wheatsequences (indicated as TaCslH1-1pro, 1-2pro and 1-3pro) in BioEditusing the Muscle comparison programme. The alignment position isnumbered above the sequences and there is a single dash (indicating agap) in the barley sequence introduced to optimise the alignment. Aminoacids are shown in their single letter form and those identical to thebarley sequence (HvCslH1(Him) are indicated by dots.

FIG. 19 shows the results of semi quantitative RT-PCR and Q-PCRexpression analysis of the barley cv. Himalaya CslH1 gene duringcoleoptile development. Panel A shows semi quantitative RT-PCR showingthe expression pattern of the barley CslH1 gene during growth of thecoleoptile and in young leaf (L), root (R) and mid stage endosperm (E).A constitutively expressed gene (alpha tubulin) is shown as a control.Panel B shows normalized expression levels (Q-PCR) for HvCslH1 indeveloping coleoptiles at various times (days) after the initiation ofgermination.

FIG. 20 shows the results of semi quantitative RT-PCR expressionanalysis of the barley CslF and CslH1 genes during leaf development.Semi quantitative RT-PCR showing the expression pattern of the barleyCslH1 gene compared to other barley CslF genes. A constitutivelyexpressed gene (alpha tubulin) is shown as a control.

FIG. 21 shows the results of semi quantitative RT-PCR expressionanalysis of the barley cv. Himalaya and wheat CslH1 genes duringendosperm development. Semi quantitative RT-PCR showing the differencein expression pattern of the CslH1 gene in the developing endosperm ofbarley cv. Himalaya gene (upper panel) compared to wheat cv. Westonia(lower panel). DPA=days post anthesis.

FIG. 22 shows the results of quantitative RT-PCR expression analysis ofthe barley cv. Himalaya and wheat CslH1 genes during endospermdevelopment. Quantitative real time RT-PCR showing the difference inexpression pattern of the barley CslH1 gene compared to wheat CslH1 genein developing endosperm. The Ta0 dpa sample has been set to one and theother expression levels are relative to this.

FIG. 23 shows a plasmid map of the plant transformation vector used toexpress the barley cv. Himalaya CslH1 genomic sequence under control ofthe Bx17 promoter. A schematic representation of the planttransformation vector designated pZLBx17HvgH1. The boxes inside thecircular plasmid represent various genetic elements: Bx17prom=Bx17promoter driving expression of the barley HvCslH1 genomic sequence;Hvg9185_(—)1=HvCslH1 genomic clone number 1 isolated with primer pairSJ91 and SJ85; Nos3′=nopaline synthase polyadenylation sequence;NPTII=bacterial kanamycin resistance gene. The position of selectedrestriction sites is indicated outside of the plasmid map.

FIG. 24 shows a plasmid map of the plant selectable marker plasmidconferring kanamycin resistance. A schematic representation of the planttransformation vector designated pCMSTLSneo. The boxes inside thecircular plasmid represent various genetic elements: 35Sprom=CaMV 35Spromoter driving expression of the plant selectable marker gene;NPTII=plant kanamycin resistance gene; STLS intron=Solanum tuberosumlarge subunit intron; 35S polyA=CaMV 35S polyadenylation sequence; Ampres=bacterial ampicillin resistance gene. The position of selectedrestriction sites is indicated outside of the plasmid map.

FIG. 25 shows the beta glucan contents of single wheat grains from T0plant line 10 expressing the barley cv. Himalaya CslH1 gene. Graphshowing beta glucan content of individual wheat grains from a T0 linenumber 10. Beta glucan is given as a percentage of flour weight.

FIG. 26 shows a quantitative RT-PCR expression analysis of CslH1 genesin empty vector control (208) and transgenic (236) barley. Expression isshown in leaf and developing grain at 7 days after pollination (7D) and14 days after pollination (14D).

FIG. 27 shows a comparison of the DNA coding sequence and amino acidsequence identity/similarity for barley and wheat CslH sequences.HvCslH1=DNA coding sequence from barley cv. Schooner (SEQ ID NO: 1) andcorresponding amino acid sequence (SEQ ID NO: 2); HvCslH1 (Him) DNAcoding sequence from barley cv. Himalaya (SEQ ID NO: 69) andcorresponding amino acid sequence (SEQ ID NO: 70); TaCslH1-1=DNA codingsequence from wheat cv. Chinese Spring (SEQ ID NO: 72) and correspondingamino acid sequence (SEQ ID NO: 75); TaCslH1-2=DNA coding sequence fromwheat cv. Chinese Spring (SEQ ID NO: 73) and corresponding amino acidsequence (SEQ ID NO: 76); TaCslH1-3=DNA coding sequence from wheat cv.Chinese Spring (SEQ ID NO: 74) and corresponding amino acid sequence(SEQ ID NO: 77).

EXAMPLE 1 Barley has Only One CSLH Gene

Candidate CSLH genes in barley were initially identified by queryingonline EST databases, such as the discontinued Stanford cell wallwebsite, NCBI, HarvEST, GrainGenes, Barley Gene Index and BarleyBase,with rice CSLH sequences. All CSLH-related ESTs from barley could bealigned into a single contiguous sequence of ˜1,500 bp that included theentire 3′ untranslated region (UTR) and a region encoding theCOOH-terminal 488 (of an expected ˜750) amino acid residues of theprotein (Table 2). This gene was designated HvCslH1. Screening of abarley BAC library with HvCslH1-derived probes identified severalgenomic clones all containing HvCslH1, from which the missing 5′ end wasobtained (data not shown). A 2,430 bp HvCslH1 cDNA fragment wasPCR-amplified, contains a single 2,256 bp ORF, and encodes a proteinwith a predicted MW of 82.6 kDa and a pI of 7.0 (FIG. 6A). Analysis ofthe conceptual translation of this sequence with ARAMEMNON found betweenfive and nine transmembrane domains (TMDs), with the consensus among thedifferent programs being two NH₂-terminal and four COOH-terminal TMDs(FIG. 6B) and both termini of the mature protein predicted to becytoplasmic. This topology also places the large, central domaincontaining the D,D,D,QFKRW motif within the cytoplasm (FIG. 6C). At thenucleotide level, HvCslH1 shares 68-74% identity (62-69% amino acididentity) (see Example 6) with the three rice CSLH genes (Hazen et al.,Plant Physiol 128: 336-340, 2002). A phylogenetic reconstruction showsHvCslH1 to be the likely barley ortholog of OsCSLH1 (FIG. 7). Geneticmapping of HvCslH1 using a Sloop×Halcyon doubled haploid population(Read et al., Aust J Agr Res 54: 1145-1153, 2003) showed that HvCslH1 ison the short arm of chromosome 2H, approximately 1.5 cM from a clusterof four HvCSLF genes (HvCSLF3, 4, 8, 10) that Burton et al. (PlantPhysiol 146: 1821-1833, 2008) reported was within a major QTLcontrolling β-glucan content in ungerminated barley grain (Han et al.,Theor Appl Genet 91: 921-927, 1995; FIG. 8).

Supporting Information

A BAC library screening was employed to obtain a complete set offull-length HvCslH family members. BAC filters containing 6.5equivalents of the barley genome (cv. Morex) were screened and threeclearly positive clones identified (data not shown). When a blot of BACDNA from these clones digested with Hind III was probed, the same threeclones, 3-5-10, 3-7-3 and 3-7-8, were verified as being positive. Thedigestion pattern of BACs 3-5-10 and 3-7-8 appeared identical and manybands were common to BAC 3-7-3, indicating that all 3 BACs coveridentical or very similar regions of the barley genome. When a genomicDNA blot was hybridised with the same probe, single bands were observedin lanes digested with Hind III, Eco RI or Eco RV, corroborating the BACdigestion results. As all HvCslH ESTs are also derived from a singlegene (Table 2), these data strongly suggest that there is only one CSLHgene in the barley genome.

An adaptor primer PCR method (Siebert et al., Nucl Acids Res 23:1087-1088, 1995) was used to identify the 5′ end of HvCslH1. DNA wasisolated from BACs 3-5-10 and 3-7-3, digested with a range ofrestriction enzymes producing blunt-ended DNA fragments to whichadaptors were ligated. Nested PCR was then performed with adaptor- andHvCslH1-specific primers (Table 3) in order to amplify fragmentscontaining the 5′ end of the gene. Amplification of BAC 3-7-3 DNAdigested with Nru I using primers AP2 and H1R6 successfully amplified a1.3 kbp fragment that contained all but ˜20 amino acids of theN-terminal sequence. Direct sequencing of BAC 3-7-3 DNA with the H1R10primer, an antisense primer designed to anneal near the 5′ end of the1.3 kb fragment, enabled the remainder of the open reading frame plus748 bp of upstream sequence to be identified. As predicted from earlierresults, the sequence obtained from BAC 3-5-10 was identical to BAC3-7-3, confirming that there is only one CSLH gene within the barleygenome.

TABLE 2 List of ESTs derived from HvCslH1. ESTs are listed in order ofalignment 5′ to 3′. Accession no. (5′ to 3′) Cultivar Source tissueCA013594 Barke early endosperm, 0-16 hours after imbibition BJ470984Haruna Nijo adult top three leaves at heading stage BJ471865 Haruna Nijoadult top three leaves at heading stage BJ473288 Haruna Nijo adult topthree leaves at heading stage BJ452043 Akashinriki vegetative stageleaves BJ471909 Haruna Nijo adult top three leaves at heading stageAV932844 Haruna Nijo adult top three leaves at heading stage BJ469514Haruna Nijo adult top three leaves at heading stage AV933503 Haruna Nijoadult top three leaves at heading stage AV933012 Haruna Nijo adult topthree leaves at heading stage AV932649 Haruna Nijo adult top threeleaves at heading stage AV932549 Haruna Nijo adult top three leaves atheading stage BJ475824 Haruna Nijo adult top three leaves at headingstage BJ476822 Haruna Nijo adult top three leaves at heading stageAV934650 Haruna Nijo adult top three leaves at heading stage BJ477472Haruna Nijo adult top three leaves at heading stage AV935479 Haruna Nijoadult top three leaves at heading stage AV935951 Haruna Nijo adult topthree leaves at heading stage AV832539 Akashinriki vegetative stageleaves AV936586 Haruna Nijo adult top three leaves at heading stageCB881459 Barke male inflorescences (approx. 2 mm in size), green antherstage AV934667 Haruna Nijo adult top three leaves at heading stageBJ475744 Haruna Nijo adult top three leaves at heading stage BJ459600Akashinriki vegetative stage leaves AV832391 Akashinriki vegetativestage leaves

TABLE 3 Primers used in cloning and amplifying HvCslH1 and in situ PCR analysisGene Primer name Primer sequence (5′ to 3′) Technique HvCslH1 H1F1TTGACCGGACAACGGATCC DNA blot analysis, (SEQ ID NO: 13)BAC screening, gene mapping, in situ PCR HvCslH1 H1F2 CTGGAGATACTCATCAGCNorthern blotting, (SEQ ID NO: 14) transgenic plant genomic DNA PCRscreening HvCslH1 HvCslH1cF1 TCGAGCGGTTGTTGCTTGTG HvCslH1 cDNA(SEQ ID NO: 15) amplification HvCslH1 HvH1TOPOfCACCATGGCGGGCGGCAAGAAGCTG Binary vector (SEQ ID NO: 16) constructionHvCslH1 H1R1 CGTCACCGGGATGAAAAC DNA blot analysis, (SEQ ID NO: 17)BAC screening, genome walking PCR, in situ PCR HvCslH1 H1R2TGACGCTCCACGGCATTC In situ PCR (priming (SEQ ID NO: 18) cDNA synthesis)HvCslH1 H1R5 GGCTGGCCATCGAAATATTG BAC screening, (SEQ ID NO: 19)genome walking PCR, gene mapping, in situ PCR HvCslH1 H1R6GAGCGTTGGTCATCACGG Genome walking (SEQ ID NO: 20) PCR HvCslH1 H1R7CACATCGCGTGTAGGGC Genome walking (SEQ ID NO: 21) PCR HvCslH1 H1R10CACTTGCCGTTCATGTTG Adaptor ligation (SEQ ID NO: 22) PCR, BAC sequencingHvCslH1 HvCslH1cR1 CCTGCTTGAGTCTTCGTTACATGTTC HvCslH1 cDNA(SEQ ID NO: 23) amplification HvCslH1 HvH1TOPOr CGCTTCCAATATTTCGATGBinary vector (SEQ ID NO: 24) construction, Northern blotting,transgenic plant genomic DNA PCR screening Generic Adaptor 1CTAATACGACTCACTATAGGGCTCGAG Adaptor ligation PCR CGGCCGCCCGGGCAGGT(SEQ ID NO: 25) Generic Adaptor 2 P-ACCTGCCC-NH₂ Adaptor ligation PCR(SEQ ID NO: 26) Generic AP1 GGATCCTAATACGACTCACTATAGGGCAdaptor ligation PCR (SEQ ID NO: 27) Generic AP2 AATAGGGCTCGAGCGGCAdaptor ligation PCR (SEQ ID NO: 28) 18S rRNA Hv18SRTrGTTTCAGCCTTGCGACCATACT In situ PCR (priming (SEQ ID NO: 29)cDNA synthesis) 18S rRNA Hv185f GGTAATTCCAGCTCCAAT In situ PCR(SEQ ID NO: 30) 18S rRNA Hv185r GTTTATGGTTGAGACTAG In situ PCR(SEQ ID NO: 31)

EXAMPLE 2 Expression of HvCslH1 in Arabidopsis Results in Deposition of(1,3;1,4)-β-D-Glucan

For heterologous expression in Arabidopsis, the HvCslH1 ORF was clonedinto the Gateway-enabled binary vector pGWB15 (Nakagawa et al., J BiosciBioeng 104: 34-41, 2007; FIG. 15), which placed HvCslH1 under thecontrol of the CaMV 35S promoter and added a 3×HA epitope tag to theencoded protein's NH₂-terminal end (FIG. 1A). Initial selection oftransformed Arabidopsis seeds identified a number of putative transgenicseedlings which PCR confirmed contained HvCslH1. RNA blot analysis ofthese T₁ plants showed that approximately 90% accumulated HvCslH1transcripts in rosette leaves (FIG. 1B). Western blotting using ananti-HA tag antibody was used to detect HvCslH1 protein in these lines(FIG. 1C). A mixed microsomal membrane fraction (50,000-100,000×gpellet) was prepared from pooled three-week old kanamycin-resistant T2seedlings. Western blotting with the anti-HA antibody showed that onlyfour of the 28 lines containing HvCslH1 transcripts accumulated apolypeptide of the expected size (˜90 kDa) (FIG. 1C). Occasionallyproteins of higher and lower molecular mass were also detected (e.g.lane 11). The 90 kDa-protein was not observed in total protein extracts(data not shown) or in mixed-membrane fractions prepared fromuntransformed Arabidopsis plants (FIG. 1C, Col-0 lane). It is not knownwhy HA-tagged HvCslH1 accumulated in only some of the plant lines thatexpressed HvCslH1 mRNA or why no correlation was apparent betweenHvCslH1 protein levels and either HvCslH1 transcript levels (compareFIGS. 1B and C) or with the number of HvCslH1 transgenes present in aplant (data not shown), although this has been previously observed(Burton et al., Science 311: 1940-1742, 2006) Lines 8, 11, 16 and 24,which expressed the HA-tagged HvCslH1, and line 6, which did not expressdetectable levels of the protein (control), were selected for subsequentexperimental work.

Immuno-EM was used to determine whether the walls of the transgenicArabidopsis plants accumulated detectable levels of β-glucan. Thinsections of mature leaf pieces from self-pollinated progeny of lines 8,11, 16, 24 and 6 (T2 generation) were probed with a monoclonal antibodyspecific for β-glucan (Meikle et al., Plant J 5: 1-9, 1994), followed bydetection using a secondary antibody conjugated to 18 nm gold particles.Gold particles were clearly evident in walls of the HA-tagged HvCslH1positive lines 8, 11 and 16 (FIG. 2A, C, B, respectively) but not in thewalls of either line 24, which also expressed HvCslH1 (data not shown),or line 6 (FIG. 2E) which had no detectable HvCslH1 protein. Eachpositive transgenic line showed a different pattern of tissue labeling.In line 8, patchy labeling was observed in the walls of epidermal cellsand occasionally in xylem walls (FIG. 2A) whereas in line 11, epidermaland vascular tissue walls were only lightly labelled, but heavier(albeit more patchy) labeling was observed in mesophyll walls (FIG. 2C).Broadly distributed, light labeling was present in all walls of themature leaf of line 16 (FIG. 2B). Irregular and inconsistent patterns ofectopic polysaccharide production by transgenic Arabidopsis linesexpressing genes driven by the “constitutively”-expressed 35S promoterhave been observed previously (Burton et al., 2006, supra). No labelingwas seen in leaf sections of untransformed Arabidopsis (FIG. 2D).Reduced levels of labeling were seen in leaf sections of transgenicplants that had been pre-incubated with a Bacillus subtilisendo-hydrolase which specifically hydrolyses this β-glucan (Burton etal., 2006, supra; data not shown).

To provide biochemical confirmation of the presence of β-glucan intransgenic Arabidopsis walls and to examine the fine structure of thenascent β-glucan, leaf and/or stem material was pooled from theself-pollinated T3 and T4 progeny of lines derived from plants 8, 11 and16. These lines were homozygous for the HvCslH1 transgene. Becauseβ-glucan was found to accumulate with plant age, samples were taken whenplants were in senescence. Walls were prepared and digested with a(1,3:1,4)-β-glucan-specific endo-hydrolase and the releasedoligosaccharides profiled by HPAEC and MALDI-TOF MS.(1,3;1,4)-β-D-Glucan endo-hydrolase specifically hydrolyses(1,4)-β-glucosidic linkages when these linkages are on the reducing-endside of a (1,3)-β-D-glucosyl residue. The action of this enzyme yields aseries of oligosaccharides with different degrees of polymerization(DP). The diagnostic oligosaccharides in this series are thetrisaccharide G4G3G_(R) and the tetrasaccharide G4G4G3G_(R) (where G isβ-D-glucopyranose, 3 and 4 indicate (1,3) and (1,4) linkages,respectively, and G_(R) refers to the reducing terminal residue).Variable quantities of G4G3G_(R) and G4G4G3G_(R) were released whenwalls prepared from leaf or leaf and stem from lines 8 and 11 and twoindependent lines derived from plant 16 (lines 16-1 and 16-2) weretreated with (1,3;1,4)-β-D-glucan endo-hydrolase (FIGS. 3, 9A). Theseoligosaccharides were not detected in the no-enzyme treatment control.The ratio of G4G3G_(R) to G4G4G3G_(R) (DP3:DP4) was estimated to be 3.5in line 16-1, which is similar to the DP3:DP4 ratio of 3.6 obtained forβ-glucan from the barley leaf sample. A peak that co-eluted withlaminaribiose, a (1,3)-β-linked disaccharide of glucose (G3G_(R)), wasalso observed in lines 8, 11 and 16-2 at varying levels across samples(FIG. 9A, data not shown). This product was absent from the barley andno-enzyme treatment control samples (FIG. 9A), verifying its appearanceis not due to a contaminating enzyme in the (1,3;1,4)-β-D-glucanendo-hydrolase preparation or to endogenous disaccharide or enzymeactivity within Arabidopsis. The identities of oligosaccharides in thisprofile were further confirmed by MALDI-TOF MS analysis, which showedthe presence of Hex₂, Hex₃ and Hex₄ in ratios similar to those observedin the HPAEC profile (FIG. 9B). The amounts of β-glucan in lines 16-1and 16-2, as estimated from the areas of the G4G3G_(R) peaks, were0.005% and 0.003% (w/w) of total wall, respectively.

EXAMPLE 3 HvCslH1 is Located in ER- and Golgi-Associated Vesicles butnot the Plasma Membrane of Transgenic Arabidopsis Plants ExpressingHvCslH1

Sections of high pressure-frozen leaves from line 11 were incubated withthe gold-labelled anti-HA antibody to determine the sub-cellularlocation of HvCslH1. Labelling was seen in the endoplasmic reticulum andin Golgi-derived vesicles but not in the plasma membrane (FIG. 4A, B).Similar results were observed in labelled sections of roots andseedlings (data not shown).

EXAMPLE 4 HvCslH1 is Transcribed in Barley at Low Levels in DevelopingGrain, Floral Tissues and Cells of the Leaf Undergoing Secondary CellWall Thickening

The levels of HvCslH1 transcripts in various barley tissues weredetermined using quantitative RT-PCR (QPCR). The gene-specific primersare presented in Table 4.

TABLE 4  List of primers used in Q-PCR analysis. PCR primers and PCRproduct sizes are given in base pairs,together with optimal acquisition temperaturesfor genes analysed. Hv, Hordeum vulgare. Forward Primer (5′-3′)PCR product Acquisition Gene Reverse Primer (5′-3′) (bp) Temp. (° C.)Hv GAPDH GTGAGGCTGGTGCTGATTA 198 80 (SEQ ID NO: 32)CGTGGTGCAGCTAGCATTTGAGAC (SEQ ID NO: 33) Hv CyclophilinCCTGTCGTGTCGTCGGTCTAAA 122 79 (SEQ ID NO: 34) ACGCAGATCCAGCAGCCTAAAG(SEQ ID NO: 35) Hv α-Tubulin AGTGTCCTGTCCACCCACTC 248 80 (SEQ ID NO: 36)AGCATGAAGTGGATCCTTGG (SEQ ID NO: 37) Hv HSP70 CGACCAGGGCAACCGCACCAC 10883 (SEQ ID NO: 38) ACGGTGTTGATGGGGTTCATG (SEQ ID NO: 39) Hv EL1aGGTACCTCCCAGGCTGACTGT 164 80 (SEQ ID NO: 40) GTGGTGGCGTCCATCTTGTTA(SEQ ID NO: 41) HvCslH1 TGCTGTGGCTGGATGGTGTT 295 82 (SEQ ID NO: 42)GCTTTATTATTGAGAGAGATTGGGAGA (SEQ ID NO: 43)

FIG. 5 (A-C) shows that across a set of barley vegetative and floraltissue cDNAs, HvCslH1 transcripts were accumulated to levels that wereroutinely less than 1,000s copies/μl cDNA. This value is lower than someof the other barley CESAs and CSLs we have studied where values aretypically in the range of 10,000s and 100,000s copies/μl cDNA. Levels ofHvCslH1 transcripts were relatively low in tissues comprising rapidlyelongating cells, including coleoptile and leaf base, which are thosethat are actively synthesising β-glucan.

The highest levels of HvCslH1 transcripts were in leaf tip, where cellsare no longer actively growing and less β-glucan accumulates (FIG. 5C;2, 4). HvCslH1 transcription in leaf was characterised further usingRNAs isolated from six zones within the ˜13 cm-long leaves of 10 day-oldseedlings, starting from the leaf tip. These zones comprises fullymature cells (zone A), to the leaf base comprising dividing cells (zoneF). In situ PCR (see Example 5) was used to identify those cells in theleaf tip that contained the HvCslH1 mRNA. In this technique, cells inwhich gene transcripts are detected stain purple to dark brown (FIG. 5D,18S RNA positive control). Cells where no transcription is detectedstain light brown, as in the negative control (FIG. 5E). HvCslH1 wasmostly transcribed in cells that are undergoing secondary wallthickening, such as interfascicular sclerenchymal fibre and xylem cells(FIG. 5F). Immuno-EM using sections taken from barley leaf and probedwith the β-glucan antibody identified β-glucan in the walls of thesecells.

HvCslH1 transcript levels were also investigated in more detail in a24-day developing endosperm series (FIG. 5B). HvCslH1 expression was lowthroughout the starchy endosperm during development. Maximum transcriptlevels were reached at 4 DPA, approximately 1 day before β-glucan isfirst detected in endosperm walls. This transcription pattern is similarto that of several barley CSLF genes (HvCSLF3, 4, 7, 8 and 10) that arealso expressed in developing grain, although HvCSLF9 and 6 show muchhigher transcript levels.

EXAMPLE 5 Discussion

The data presented here indicate that the product of HvCslH1, a memberof the grass-specific CSLH gene family, mediates β-glucan biosynthesisin Arabidopsis. Barley appears to have only a single CSLH gene based onEST database analyses, genomic DNA blot analysis and BAC libraryscreening. EST analyses of other grasses such as bread wheat, Loliummultiflorum, Festuca arundinacae and Brachypodium distachon (allsubfamily Pooideae) have one identified CSLH gene, similar to barley,whereas maize, sorghum and sugar cane (all subfamily Panicoideae), likerice (subfamily Ehrhartoideae), appear to have multiple CSLH genes. Whenan epitope-tagged version of the HvCslH1 cDNA was heterologouslyexpressed in Arabidopsis, three of four plant lines in which protein wasdetected accumulated a polysaccharide in their walls that was recognizedby a β-glucan-specific monoclonal antibody. When isolated walls of thetransgenic lines were digested with a specific (1,3;1,4)-β-D-glucanendo-hydrolase, the characteristic trisaccharide (G4G3G_(R)) andtetrasaccharide (G4G4G3G_(R)) were detected at ratios similar to thosefound in β-glucans from barley endosperm, demonstrating that the wallsfrom the transgenic Arabidopsis lines contained β-glucan. Furthermore,epitope-tagged HvCslH1 was found in the endoplasmic reticulum and inGolgi-derived vesicles in cells of transgenic plants. The morphologicalphenotype of the transgenic Arabidopsis lines that expressed HvCslH1appeared identical to wild-type plants.

Although the overall proportion of (1,3)- and (1,4)-β-glucosyl linkagesand the ratios of the G4G3G_(R) and G4G4G3G_(R) products from(1,3;1,4)-β-D-glucan endo-hydrolase digestion of walls derived fromplant line 16-1 was similar to those observed in β-glucans isolated frombarley tissues, one unusual feature that was observed was that the majoroligosaccharide released by (1,3;1,4)-β-D-glucan endo-hydrolase from thewalls of line 16-2 was laminaribiose (G3G_(R); FIG. 9A). The presence ofG3G_(R) in variable levels was also associated with increased levels oftrisaccharide relative to the tetrasaccharide and, thus increasedDP3:DP4 ratios. The presence of G3G_(R) in wall digests of the majorityof plant lines indicates a polysaccharide containing sections ofalternating (1,3)-β- and (1,4)-β-linked glucosyl residues (-G3G4G3G4-).It is not known if these reside in a separate polysaccharide orconstitute a portion of a β-glucan chain that also has the usual finestructural features. Alternating (1,3)-β-D-glucosyl and (1,4β-D-glucosyl residues are not common in barley and other cerealβ-glucans, but do represent a significant component of the β-glucan fromthe non-flowering plant Equisetum and are also detected in β-glucansfrom a number of fungi, including basidiomycetes and ascomycetes. It ispossible that G3G_(R) arises through misregulation of the β-glucansynthase in transgenic Arabidopsis, possibly because its membranemicro-environment is different or because an unknown factor that inbarley suppresses (1,3)-β glucosidic linkage formation (or alternativelypromotes (1,4)-β glucosidic linkage formation) is present at suboptimallevels in Arabidopsis. Minor variations in the level of this factoramong the lines derived from plant 16 would account for the differentstructures that were obtained. Another possible explanation for thestructural variability in the β-glucan may relate to subtle differencesin post-assembly processing (see also Supporting Information below).

In barley, HvCslH1 was most highly transcribed in leaf tips, a tissuecomprising fully mature cells. There is no evidence to indicatecoordinate transcription of HvCslH1 and any of the barley CSLFs,suggesting that their encoded products are not components of a proteincomplex. HvCslH1 transcription, for example, was not high in elongatingcells such as the coleoptile or developing endosperm, which in barleyare the tissues where β-glucan is characteristically accumulated.Although usually found in primary cell walls of vegetative tissues whereit is implicated in the control of cell expansion and possibly as atemporary store of glucose that can be mobilized as an energy source inthe dark, β-glucan has also been found in the lignified cell walls ofxylem tracheary elements and sclerenchyma fibres, where immuno-EM usingthe antibody to β-glucan shows labeling in both the middle lamellaregion (primary wall) and secondary wall of sclerenchyma cells. Becausein situ PCR showing transcription of the HvCslH1 gene in the leaf wasrestricted to cells such as interfascicular sclerenchymal fibre andxylem cells, we suggest that a major role of this gene is in β-glucansynthesis during secondary wall development, although we cannot excludea role in primary wall β-glucan synthesis elsewhere in the plant.

Regardless of how the fine structures of β-glucans are generated, it isclear that the CSLHs can mediate the synthesis of β-glucan inArabidopsis, a finding that has implications for our understanding ofhow this polysaccharide is synthesised. Any mechanism(s) beingconsidered for the assembly of β-glucan must account for the synthesisof the predominant cellotriosyl and cellotetraosyl units, the randomlinking of these (1,4)-β-units together by single (1,3)-β-linkages andthe means by which the molar ratio of tri- to tetra-saccharide units isregulated. At least two glycosyltransferase activities might act inconcert: one that processively adds (1,4)-β-linked glucose residues toassemble the tri- and tetra-saccharides and the other that adds single(1,3)-β-linkages. Based on our current knowledge of polysaccharidesynthases several mechanisms are hypothetically possible. The simplestexplanation is that the one polypeptide is responsible for the synthesisof both glucosidic linkage types. Our transgenic experiments indicatethat CSLH proteins are independently able to make a β-glucan and couldtherefore conceivably make both types of β-linkages. The CSLH family isclassified by the Carbohydrate Active Enzymes (CAZy) database as membersof glycosyltransferase family 2 (GT2) (http://www.cazy.org; Coutinho etal., J Mol Biol 328: 307-317, 2003), a family that includes enzymescapable of independently catalyzing the synthesis of either (1,3)-β- or(1,4)-β-linkages but also examples of bifunctional enzymes, i.e. enzymesthat can synthesize two types of glycosidic linkages. For example,hyaluronan synthases (HAS) synthesize a repeating disaccharide of(1,4)-β-glucuronic acid-(1,3)-β-N-acetylglucosamine units and bothtransferase activities reside in the one polypeptide. In mouse HAS1, theregion that includes the D,D,D,QXXRW motif is responsible for both theseactivities. The active site of the CSLHs, also containing theD,D,D,QXXRW motif, might be similarly bifunctional. Another possibilityis that the CSLHs synthesise only one type of glucosidic linkage withanother glucosyltransferase, common to monocots and dicots, responsiblefor synthesis of a second linkage.

EXAMPLE 6 Materials and Methods Binary Vector Construction and PlantTransformation

The HvCslH1 ORF was amplified from barley cv. Schooner mature leaf tipcDNA with Herculase® (Stratagene) using primers HvH1TOPOf and HvH1TOPOr(Table 3) and the PCR product cloned into pENTR/D-TOPO (Invitrogen).Using the manufacturer's protocol (Invitrogen), an LR reaction was usedto clone the cDNA into the destination vector pGWB15 containing anNH₂-terminal 3×HA tag (Nakagawa et al., J Biosci Bioeng 104: 34-41,2007) and the predicted sequence confirmed by DNA sequencing. TheHvCslH1::pGBW15 construct was transferred from Escherichia coli intoAgrobacterium tumefaciens strain AGL1 via triparental mating using thehelper plasmid pRK2013. Arabidopsis thaliana Col-0 plants weretransformed using the floral dip method (Clough and Bent, Plant J 16:735-743, 1998).

RNA Blot Analysis

Samples of ˜10 μg total RNA extracted from mature rosette leaves of T1plants using TRIzol® (Invitrogen) were prepared and separated on a 1%w/v agarose-formaldehyde gel (Farrell, RNA methodologies: A laboratoryguide for isolation and characterization, Academic Press, Inc., SanDiego, 1993). RNA was transferred to Hybond™ N⁺ membranes,pre-hybridised and hybridised according to the method outlined in theGene Images CDP-Star detection module (Amersham-Biosciences). A HvCslH1fragment amplified with primers H1F2 and HvH1TOPOr (Table 3) was labeledusing the Gene Images Random Prime labeling module (Amersham) followingthe manufacturer's protocol and used as the probe.

Quantitative PCR Analysis

RNA extractions, cDNA syntheses and QPCR were carried out as describedin Burton et al. (Science 311, 1940-1942, 2006; Plant Physiol 134,224-236, 2004) with the modifications listed in Burton et al. (PlantPhysiol 146, 1821-1833, 2008). The primer sequences of the barleycontrol genes are listed in Table 4.

In Situ PCR

In situ PCRs were conducted according to the method of Koltai & Bird(Plant Physiol 123: 1203-1212, 2000) with the following modifications.After tissue sectioning, genomic DNA was removed by treatment for 6 h at37° C. in 1× DNase buffer and 4 U RNase-free DNase (Promega). cDNAsynthesis was carried out using Thermoscript™ RT (Invitrogen) exceptthat the RNase H step was omitted and a gene-specific primer (1 μg,Table 3) used for reverse transcription. PCRs were carried out in afinal volume of 50 μL containing 1×PCR buffer, 200 μm dNTPs (Promega),0.2 nmol digoxigenin-11-dUTP (Roche), 2 mM MgCl₂, 200 ng of each primerand 2 U Taq DNA polymerase (Invitrogen). Cycling parameters were asfollows: initial denaturation at 96° C. for 2 min, then 40 cycles of 94°C. for 30 sec, 59° C. for 30 sec, 72° C. for 1 min. Sections were thenwashed, incubated with 1.5 U alkaline phosphatase-conjugatedanti-digoxigenin Fab fragments (Roche) and developed for 10-20 min asoutlined by Koltai & Bird (2000, supra). For negative control sections,reverse transcriptase was omitted and all the Hv 18S rRNA primersincluded to check whether there was any amplification from genomic DNA.

Preparation of Mixed Microsomal Membranes

T1 seed of HvCslH1 transgenic plants was collected and ˜100 seeds sownonto 1×MS agar media containing 50 mg/L kanamycin (Sigma). After 3weeks, kanamycin-resistant seedlings were pooled, frozen in liquid N₂and ground at 4° C. in a mortar and pestle containing homogenisingbuffer (50 mM NaPO₄ buffer, pH 7.5, 0.5 M sucrose, 20 mM KCl, 10 mM DTT,0.2 mM PMSF, 83 μL plant protease inhibitor cocktail (Sigma, P9599)).Homogenate was filtered through a 50 μM mesh and the S/N centrifuged at6,000×g for 10 min at 4° C. The S/N was decanted and centrifuged at50,000×g for 30 min at 4° C. in 4.5 ml ultracentrifuge tubes (Beckmann).The 50,000×g S/N was decanted and the pellet resuspended in 10 mMTris-MES buffer, pH 7.5 using a glass-teflon homogenizer. Theresuspended pellet was diluted to 4.5 mL with Tri-MES buffer andcentrifuged at 100,000×g for 1 h at 4° C. The pellet was resuspended in0.25 M sucrose, 10 mM Tris-MES buffer, pH 7.5, as outlined above.Protein concentration was measured using Bradford assay reagent (BioRad)using bovine serum albumin as the standard.

Western Blotting

Samples of membrane protein (30 μg) were incubated at 60° C. for 20-60min in 200 mM dithiothreitol and sample buffer (37.5 mM Tris-HCl, pH7.0, 10% glycerol, 3% sodium dodecylsulphate (SDS), 0.025% bromophenolblue) to give an SDS:protein ratio of 1.5 mg SDS to 30 μg protein beforeloading onto an 8% SDS-PAGE gel. After electrophoresis, gels wereblotted onto nitrocellulose (OSMONIC™ Nitropure 22 μm) in Towbin buffer(25 mM Tris base, 192 mM glycine, 20% methanol) containing 0.05% SDS at100 V for 90 min at 4° C. Membranes were then blocked overnight inTris-buffered saline (TBS; 20 mM Tris base, 150 mM NaCl) containing 3%w/v milk powder before incubation for 1 h at RT in rat anti-HApolyclonal antibody (Roche) diluted 1:1000 in TBS containing 1% BSA.Membranes were washed 3× in TBS containing 0.05% SDS (TBST), thenincubated in anti-rat IgG HRP-conjugated antibody (Dako) diluted 1:1000in TBS containing 3% w/v nonfat milk powder. Membranes were washed 3× inTBST before signal was detected with the SuperSignal® West Picochemiluminescent substrate (Pierce).

Immuno-Electron Microscopy

Arabidopsis tissues were fixed and labeled withanti-(1,3;1,4)-β-D-glucan specific antibody (Meikle et al., Plant J 5:1-9, 1994) according to Burton et al. (Science 311, 1940-1942, 2006).For labeling with anti-HA antibody, plant tissue was placed between twocopper planchets and rapidly frozen in a Leica EM high pressure freezer(set at 2.7×10⁵ kPa and at an approximate rate of −10,000° C. s⁻¹). Theplanchets were transferred into 100% ethanol in a Leica automatedfreeze-substitution unit set at −50° C. for 72 h. Samples were broughtto room temperature (RT) overnight, removed and infiltrated with LRWhite resin and embedded in gelatin capsules as detailed in Burton etal. (2006, supra). Thin sections of embedded leaf tissue were collectedon formvar-coated gold grids and incubated in a 1:200 dilution of therat anti-HA polyclonal antibody in phosphate buffered saline (PBS; 137mM NaCl, 10 mM NaPO₄, 2.7 mM KCl, pH 7.4) containing 1% w/v BSA for 1 hat RT and then overnight at 4° C. Grids were washed several times inPBS, then incubated in a 1:20 dilution of anti-rat secondary antibodyconjugated to 18 nm gold (Jackson ImmunoResearch) in PBS containing 1%w/v BSA for 1 h at RT. The grids were then washed, post stained andviewed under the TEM as described by Burton et al. (2006, supra)

Preparation of Cell Wall Material

Alcohol insoluble residue (AIR) was prepared by grinding plant materialin liquid N₂ using a mortar and pestle. Five volumes of 80% ethanol wasadded to the homogenate prior to mixing by rotation for 1 h at 4° C.After centrifugation at 3,400×g for 5 min, the supernatant was removedand the residue was refluxed twice at RT in 80% ethanol for 1 h,followed by refluxing in 50% ethanol twice for 1 h. The ethanol-solublefraction was removed and the AIR was washed once in 100% ethanol priorto drying at 40° C. under vacuum.

(1,3;1,4)-β-D-Glucan Specific Endo-Hydrolase Digestion

AIR (100 mg, prepared as described above) was incubated in 5 mL 20 mMNaPO₄ buffer, pH 6.5 for 2 h at 50° C. with continuous mixing in anincubator with shaking at 200 rpm. After 2 h, the suspension wascentrifuged (3,400×g, 5 min) and the supernatant (S/N) removed. Another5 mL of buffer was added and the incubation and centrifugation repeated.The S/N from this second incubation was used as the no enzyme negativecontrol. The pelleted AIR was resuspended in 5 mL NaPO₄ buffer to which100 μl (1,3;1,4)-β-D-glucan endo-hydrolase (McCleary et al., J Inst Brew91: 285-295, 1985) was added. The mixture was incubated for 2 h at 50°C. with continuous mixing after which the S/N was collected as the(1,3;1,4)-β-D-glucan endo-hydrolase-released oligosaccharides. Thenegative control and (1,3;1,4)-β-D-glucan endo-hydrolase-treated S/Nswere desalted on a graphitised carbon cartridge as described by Packeret al. (Glycoconj J 15: 737-747, 1998) and dried.

HPAEC Analysis

The dried (1,3;1,4)-β-D-glucan endo-hydrolase-released oligosaccharideswere dissolved in 100 μL Milli H₂O and 20 μL injected onto a CarboPacPA1 column (Dionex) equilibrated with 50 mM NaOAc in 0.2 M NaOH using aDionex BioLC ICS 300 system (Dionex) equipped with a pulsed amperometricdetector (PAD) and autosampler. Oligosaccharides were eluted at 1 mL/minwith a linear gradient of NaOAc from 50 mM in 0.2M NaOH to 350 mM in 0.2M NaOH over 15 min. Laminaribiose (Seigaku), maltose and cellobiose(both from Sigma) were run as standards.

MALDI-TOF MS Analysis

Aliquots (30 μL) of the remaining (1,3;1,4)-β-D-glucanendo-hydrolase-released oligosaccharides were lyophilised, dissolved inDMSO and methylated using the NaOH method (Ciucanu and Kerek, CarbResearch 131: 209-217, 1984). Methylated oligosaccharides werepartitioned into dichloromethane (DCM) and the DCM phase washed 3× withMilliQ water. The DCM phase was dried under a N₂ stream beforere-dissolving in 10 μL 50% acetonitrile. A 1 μL aliquot was mixed with 1μL 2,5-dihydroxy benzoic acid matrix (10 mg/mL dissolved in 50%acetonitrile) and 1 μL of the mix was spotted onto a MALDI plate foranalysis in a MALDI TOF mass spectrometer (Voyager DSTR, AppliedBiosystems).

EST Analyses, Contig Assembly and Bioinformatics

CSLH ESTs were obtained by querying public databases including the nowdiscontinued Stanford Cell Wall website, NCBI(http://www.ncbi.nlm.nih.gov/), HarvEST (http://harvest.ucr.edu/),GrainGenes (http://wheat.pw.usda.gov/GG2/index.shtml), Barley Gene Index(http://compbio.dfci.harvard.edu/tgi/plant.html) and BarleyBase(www.barleybase.org) using the BLAST search tool (Altschul et al., NuclAcids Res 25: 3389-3402, 1997). Sequences were assembled into contigsusing either Sequencer™ 3.0 (GeneCodes) or ContigExpress, a module ofVector NTI® Advance 9.1.0 (Invitrogen). DNA or protein sequences werealigned using ClustalX (Thompson et al., Nucl Acids Res 24: 4876-4882,1997). Phylogenetic analysis was carried out using the in-builtneighbour joining algorithm and tree robustness assessed using 1000bootstrapped replicates. Sequence similarities were calculated usingMatGat 2.02 (http://bitincka.com/ledion/matgat/) (Campanella et al., BMCBioinformatics 4: 29, 2003). Transmembrane domains were predicted usingthe suite of programs described in ARAMEMNON(http://aramemnon.botanik.uni-koeln.de) (Schwacke et al., Plant Physiol131: 16-26, 2003). Motifs predicting post-translational modificationswere identified using the programs listed in ExPasy under the Tools menu(http://www.expasy.org/tools/#pattern). Protein parameters werecalculated using ProtParam at ExPasy(http://www.expasy.org/cgi-bin/protparam).

Barley BAC Screening

BAC filters containing 6.5 equivalents of the barley genome from thenon-Yd2 cv. Morex (Clemson University Genomics Institute, CUGI) wereblocked for 6 h at 65° C. in prehybidisation solution (0.53 M NaPO₄buffer pH 7.2, 7.5% w/v SDS, 1 mM EDTA, 11 μg/ml salmon sperm DNA). Theradiolabeled cDNA and gDNA fragment amplified with primers H1F1 and H1R1or H1R5 (Table 3) was added and incubated for 24 h at 65° C. Filterswere washed 3× with 2×SSC, 0.1% SDS at RT. Final washes were done with1×SSC, 0.1% SDS. Filters were exposed to X-ray film for 2 d. PositiveBAC clones were identified and ordered as directed on the CUGI website(http://www.genome.clemson.edu). Clones were streaked onto LB agarcontaining 25 μg/ml chloramphenical and grown overnight at 37° C.Colonies for each clone were picked, placed on gridded nylon membranesresting on LB agar containing 25 μg/ml chloramphenicol and incubatedovernight at 37° C. DNA was fixed to the membrane and denatured byplacing on filter paper soaked in 0.4 M NaOH for 20 min, thenneutralized by placing on filter paper soaked in neutralizing solution(1.5 M NaCl, 0.5 M Tris-HCl pH 7.2, 1 mM EDTA). Membranes were thenwashed 3× in 2×SSC, 0.1% SDS and hybridized using standard conditions(Sambrook et al., Molecular cloning: a laboratory manual, Cold SpringHarbour Laboratory Press, New York, 1989).

BAC DNA Isolation

Positive clones were cultured overnight in LB broth containing 25 μg/mlchloramphenicol at 37° C. Cells were pelleted by centrifugation(12,000×g, 3 min) and the pellet resuspended in 90 μL TES buffer (25 mMTris-HCl pH 8.0, 10 mM EDTA, 15% w/v sucrose). An aliquot (180 μL) oflysis solution (0.2 M NaOH, 1% SDS) was added and mixed gently, followedby 135 μL 3 M NaOAc pH 4.6. The chromosomal DNA was pelleted bycentrifugation (12,000×g, 15 min). The S/N was collected and 2 μL RNaseA (10 mg/mL) added and incubated for 1 h at 37° C. A 400 μL aliquot ofTris-saturated phenol-chloroform (1:1 ratio) was added and the samplescentrifuged again (12,000×g, 5 min). The S/N was collected and BAC DNAprecipitated using 2-3 volumes chilled 95% ethanol for 10 min at RT. TheBAC DNA was pelleted by centrifugation (15,000×g, 15 min), washed in 70%ethanol, resuspended in 20-50 μL TE and stored at 4° C.

Genome Walking

The adaptor ligation method of Siebert et al. (Nucl Acids Res 23:1087-1088, 1995) was used to amplify fragments of genomic DNA upstreamof known CSLH EST sequence. Restriction enzymes used to digest barleygenomic DNA were Eco RV, Nru I, Pvu II, Sca I or Ssp I. Primary PCRreactions were conducted in 25 μL volumes containing 2 μL ligated DNA(1:10 dilution), 1×PCR buffer, 2 mM MgCl₂, 100 ng each of adaptor primerAP1 and antisense primer H1R7 (Table 3), 0.4 mM dNTPs and 1 unit Taqpolymerase (Invitrogen). Cycle parameters were as follows: 96° C. for 2min then 40 cycles of 94° C. for 30 sec, 59° C. for 30 sec, 72° C. for 1min, and a final step at 72° C. for 7 min. A secondary PCR reaction wasconducted with 1 μL of the primary PCR using 100 ng each of adaptorprimer AP2 and the nested primer H1R6. Reaction composition and cycleparameters were the same as above except that an annealing temperatureof 61° C. was used.

BAC Sequencing

For sequencing, between 0.5 and 1 μg of isolated BAC DNA was combinedwith 5 pmol primer and 1× Big Dye Terminator v 3.1 (BDT) mix (AppliedBiosystems, USA) in a final volume of 20 μL. Cycle parameters were asfollows: 96° C. for 15 min, then 65 cycles of 96° C. for 10 sec, 55° C.for 10 sec and 60° C. for 4 min. DNA was precipitated with 0.1 vol 3MNaOAc pH 5.2 and 2.5 vol 95% ethanol on ice for 10 min, then pelleted byspinning at 12,000×g for 30 min. The pellet was rinsed with 70% ethanol,dried and sent to AGRF (Brisbane, Australia) for sequencing.

Mapping of HvCslH1

Genetic mapping was done using a Sloop×Halcyon doubled haploid (DH)mapping population of 60 lines (Read et al., Aust J Agric Res 54:1145-1153, 2003). Using standard methods of DNA blot hybridization(Sambrook et al., 1989, supra) a HvCslH1 probe PCR-amplified usingprimers H₁F₁ and H1R5 (Table 3) was hybridized to membranes containingparental line genomic DNA digested with one of six restriction enzymes(Bam HI, Dra I, Eco RI, Eco RV, Hind III, Xba I). The dihybridpopulation was then digested with enzymes that gave a clear polymorphism(Dra I). Polymorphisms were scored and HvCslH1 map location determinedusing the ‘find best location’ function of MapManager QT version 0.30(Manly et al., Mamm Genome 12: 930-932, 2001). Map locations werecorrelated with QTL data using resources available athttp://www.barleyworld.org/.

Arabidopsis Growth Conditions

Arabidopsis seeds were surface-sterilized in a sterilization solution(sodium hypochlorite (2% available chlorine), drop of Tween-20) for 15min then rinsed 4× with sterile MilliQ water.

Surface-sterilized seed was spread onto 85×25 mm Petri dishes containing50 mL of sterile 1×MS medium (4.33 g/L Murashige and Skoog basal salts(Phytotechnology Laboratories), 2% w/v sucrose, 1% w/v bactoagar). Forselection of transformants, 50 mg/L kanamycin (Sigma) was added to themedium. Plates were placed in a cold room for 3-5 days at 4° C. tosynchronize germination. Cold-stratified plates were then transferredinto controlled environment growth cabinets (Thermoline L+M model TPG1260 TO-5×400, Smithfield, NSW, Australia) with day and nighttemperatures of 23° C. and 17° C., respectively. The average lightintensity at rosette leaf level was ˜70 μE m⁻² sec⁻¹ supplied by 3-footfluorescent tubes (Sylvania Standard F30W/133-T8 Cool White) during a 16h light cycle. After 3 weeks on MS plates, individual plantlets weretransferred into hydrated 42 mm diameter Jiffy pellets. Nine rows of sixpellets were arranged in trays with three trays being housed on each2×3.5-foot wire rack shelf. Relative humidity was measured to be between60 and 70%. Plants were watered with tap water supplemented with Peter'sProfessional™ General Purpose plant fertilizer (Scotts Australia) bysub-irrigation every 2-3 days.

Genomic DNA Extraction and PCR Analysis of Arabidopsis Transgenics

DNA was extracted from a single Arabidopsis leaf according to the methoddescribed in Edwards et al. (Nucl Acids Res 19: 1349, 1991). A 1 μLaliquot of genomic DNA was used as template in PCR screens of transgenicplants using primers H₁F₂ and HvCslH1TOPOr (Table 3) with the followingcycling regime: 94° C. for 2 min followed by 35 cycles of 94° C. for 20sec, 57° C. for 30 sec, 72° C. for 30 sec.

EXAMPLE 7 Alignment of CslH DNA and Amino Acid Sequences from Rice andBarley

An alignment of the DNA and amino acid sequences for the CslH sequencesin both rice and barley was performed to calculate the percent identityand similarity between the sequences, the results of which are shown inFIG. 10. The DNA and protein sequences were aligned and compared usingthe default parameters in MatGAT version 2.02 downloaded fromhttp://bitincka.com/ledion/matgat/.

Multiple sequence alignments and phylogenetic tree generation wasperformed using the ClustalX program as described by Thompson et al.(Nucl Acids Res 25: 4876-4882, 1997). The protein alignment andresultant phylogenetic tree are shown in FIGS. 11 and 12, respectively.

EXAMPLE 8 Cross of HvCslH1 and OsCSLF2 Transgenic Arabidopsis Lines

Two transgenic Arabidopsis lines, 15-8 and 15-11, in which the taggedHvCslH1 protein was detected using an anti-HA antibody, were chosen togenetically cross with two other transgenic Arabidopsis lines containingOsCslF2, H37 and H17-4, as described by Burton et al. (Science 311:1940-1942, 2006). It was thought that by expressing the HvCslH1 andOsCSLF2 proteins in the same cell types, higher levels of(1,3;1,4)-β-D-glucan above those observed in single gene (CSLH or CSLFonly) transgenic Arabidopsis plants, could potentially be deposited intocell walls. In addition, this would aid in detecting(1,3;1,4)-β-D-glucan in immuno-electron microscopy studies as well as inchemical cell wall analyses.

All four of the parental lines were confirmed to contain(1,3;1,4)-β-D-glucan in their cell walls by immuno-electron microscopy(FIG. 13). Individuals from each of the four populations were used asmale and female parents. Flowers of the female parent (e.g. individualH37-5) were emasculated prior to anther dehiscence and pollinated usingdehisced anthers from the male parent (e.g. individual 15-8-3). Eachcrossed flower was labelled and the resulting seed pods collected upondehydration.

The progeny of each cross were sown in soil and their genotypesdetermined by PCR using leaf genomic DNA as template andHvCslH1-specific primers and, in a separate reaction, OsCslF2-specificprimers. Mature leaves were fixed, embedded, sectioned and labeled with(1,3;1,4)-β-D-glucan monoclonal antibody. A number of the progeny werefound to have greater levels of labelling than the parental lines, asobserved in FIG. 14. For example, the labelling in the epidermal cellsof the individual shown in Panel D is much heavier than compared to its15-8-3×H37-7 parents (FIG. 13). A sib with the same genotype (FIG. 14,panel C) showed consistent yet lower levels of epidermal cell walllabeling.

EXAMPLE 9 Cloning of CslH cDNA and Genomic Sequences from BarleyCultivar Himalaya and Wheat

A full length cDNA sequence of the CslH1 gene was isolated from barleycultivar Himalaya using a combination of barley EST sequences, PCR fromcDNA using primers based on the rice CslH1 gene sequence(LOC_Os10g20090) and 5′RACE.

The 2333 bp consensus sequence designated HvCslH1(Him) (SEQ ID NO: 69)is shown in FIG. 16. There is a single long open reading frame of 751amino acids (SEQ ID NO: 70).

Oligonucleotide primers SJ91 and SJ85 were designed from the 5′ and 3′ends of the consensus sequence and used to amplify a 3203 bp DNAfragment from genomic DNA designated HvCslH1gHim (SEQ ID NO: 71) in FIG.17.

Alignment of the barley cDNA sequence and genomic sequences indicatedthat the CslH gene has eight small (approximately 100 bp) introns eachflanked by the consensus GT . . . AG splice donor/acceptor sites (FIG.17).

A wheat homolog of CslH1 was identified in the TIGR database asTC255929. Three classes of sequences made up this tentative consensus asexemplified by ESTs CJ614392, CJ609729 and CJ721204. PCR primers weredesigned from the barley sequence surrounding the ATG initiation codon(SJ163) and from the consensus sequence of all three EST types at the 3′end (SJ164) and used to amplify a full length genomic fragment fromwheat cultivar Chinese Spring. Two sequence types were identified anddesignated TaCslH1-1 (SEQ ID NO: 78) and TaCslH1-2 (SEQ ID NO: 79). Thethird homeolog designated TaCslH1-3 (SEQ ID NO: 80) was isolated usingprimers SJ204 and SJ164 as described in more detail in materials andmethods.

Comparison with the barley sequences indicated that the intron-exonjunctions were conserved in all three genes (FIG. 17). The three wheatgenes are 94.8-96.1% identical.

The predicted coding region sequences of the three wheat CslH1 genes(SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74) each encode apolypeptide of 752 amino acids (SEQ ID NO: 75, SEQ ID NO: 76 and SEQ IDNO: 77).

The DNA coding sequences and amino acids sequences of the barley andwheat CSLH1 genes were aligned using the muscle alignment program andthe percentage identity and similarity was calculated using a PAM250matrix. A table showing the percentage identity and similarity is showin FIG. 27.

As shown in FIG. 27, the wheat proteins are about 94-95.0% identical toeach other and about 92.6-93.1% identical to the barley proteins.

EXAMPLE 10 CslH Gene Expression in Barley and Wheat

Expression of the CslH1 gene was examined by semi quantitative (RT-PCRand gel electrophoresis) and quantitative (real time PCR) methods.

The coleoptile is a good tissue to examine expression of genes relatedto beta glucan biosynthesis since the levels of beta glucan increase asthe coleoptile grows and then decline after growth has stopped. TheCslH1 gene shows maximum expression only after growth has ceased and ishigh in the oldest tissues (6-8 days old, as shown in FIG. 19A/B).

Other tissues were also examined. In developing leaf, the CslH1 geneshows differential and maximum expression in the oldest tissue at thetip of the leaf (FIG. 20). It appears from these results that the CslH1gene is preferentially (although not exclusively) expressed in cellsthat have stopped dividing and elongating and are thus differentiating.Cells in the maturing endosperm would be in a similar phase ofdevelopment, ie. cell division would have stopped, cell enlargementwould be slowing with the cells differentiating into specialised starchstorage parenchyma.

In barley endosperm tissue, CslH1 gene expression peaked around 4 dayspost anthesis and then increased during later stages to reach a maximumat 28 days (FIG. 21).

There was a large difference in CslH1 gene expression in wheat whereexpression peaked at 4 days post anthesis after which expression wasvery low. These results were confirmed by real time PCR which showedthat at 28 days post anthesis, the CslH gene was expressed about 10 foldhigher levels in barley than in wheat (FIG. 22).

EXAMPLE 11 Overexpression of the Barley CslH Gene in Wheat Grain

Transgenic wheat plants were generated by biolistics transformation withthe full length genomic HvCslH1 (cv. Himalaya) gene under control of theglutenin promoter such that expression should only occur in endospermtissues (FIG. 23). Lines were screened for the presence or absence ofthe transgene by PCR of young leaf material. Twelve PCR positive linesand three PCR negative lines (H1-2, -7 and -11) were grown to maturityin the glasshouse. RNA was isolated from developing grain atapproximately 15 days post anthesis and cDNA was made using SuperscriptIII. Expression of the barley transgene was then analysed by real timePCR. Table 5 shows the relative expression levels compared to theendogenous wheat CslH gene as the primers used amplify both the wheatand barley genes.

TABLE 5 Relative expression of CslH gene in wheat endosperm Line Run 1Run 2 Run 3 H1-1 468 225 H1-2 2 H1-3 206 H1-4 620 497 243 H1-5 299 411H1-6 952 H1-7 63 H1-8 140 230 H1-9 1771 H1-10 4396 4052 H1-11 26 6 H1-1210 1103 H1-14 1013 352 H1-15 10 H1-13 1 1

Most of the lines expressed the barley CslH gene at several hundred foldhigher levels than the controls with line 9, 10, 12 and 14 showing thehighest expression (greater than one thousand fold higher).

At maturity, single grains from were analysed for beta glucan contentand a summary of the results are shown in Table 6:

TABLE 6 Beta glucan content of transgenic wheat flour Average Transgenicline Beta glucan std dev Max beta glucan H1-1 0.81 0.08 .8 H1-2 0.680.02 .7 H1-3 0.89 0.05 .9 H1-4 0.82 0.05 .8 H1-5 0.83 0.21 1.1 H1-6 0.910.09 1.0 H1-7 0.65 0.05 .7 H1-8 0.87 0.15 1.1 H1-9 1.17 0.33 1.9 H1-101.12 0.39 1.9 H1-11 0.82 0.14 1.0 H1-12 1.23 0.26 1.7 H1-14 0.99 0.191.4 H1-15 0.60 0.26 .8 H1-16 1.00 0.11 1.2 PCR − (2, 7, 11) average 0.690.10 1.0 PCR + (rest) average 0.97 0.11 1.9

The PCR negative lines all had the lowest beta glucan contents averaging0.69% of grain weight, whereas grain from the PCR positive lines had anincreased average beta glucan content of 0.97%. The last column of Table6 shows the maximum beta glucan content of any single grain from a givenline—the highest PCR negative line was 1.0% (and most grains were muchlower than this) but several of the PCR positive lines had grains withsignificantly increased beta glucan levels with line 9 and line 10 (thehighest expressers) having grains with up to 1.9% beta glucan. Theselevels of beta glucan have never been seen before in wheat.

The heads from these T0 plants contain T1 seed which are segregating forthe transgene. If the DNA is inserted at a single locus a ratio of threetransgenic to one wild type seed should be observed. FIG. 25 shows thebeta glucan levels of individual T1 seeds from the H1 transgenic line 10from which it can be seen that approximately three quarters (47/61) havehigher beta glucan levels than the average of the PCR negative lines(0.7%). From the ratio of the highest beta glucan level (1.9%) to theaverage PCR negative level (0.7%) the increase in beta glucan content is2.7 times that normally seen in wild type wheat grains. A furthersignificant observation is that a high proportion of the grains have atleast 1.4% beta glucan.

It is expected that further increases in beta glucan will be seen inthese grains when the lines are made homozygous and gene dosageincreases.

EXAMPLE 12 Materials and Methods for Examples 9 to 11 Plant Material

Barley (Hordeum vulgare) cultivar Himalaya and wheat (Triticum aestivum)cultivar Chinese Spring, Westonia and Bob White26, were grown understandard glasshouse conditions.

Primer Sequences

The primer sequences referred to in Examples 9 to 11 and this exampleare shown below in Table 7:

TABLE 7  Primer sequences for Examples 9 to 12 Primer Target geneSequence (5′-3′) Sequence Identifier SJ27 CslH1 AGGCGTGGTTCGCGTTCGSEQ ID NO: 44 SJ28 CslH1 CAGCGCGTAGTACGTCAC SEQ ID NO: 45 SJ72 CslH1CAGCCGTGATGACCAACG SEQ ID NO: 46 SJ73 CslH1 GTTGCCAAAGGGATCGTCSEQ ID NO: 47 SJ79 CslH1 GCGGTCGTGACGAACATGTCCAC SEQ ID NO: 48 SJ75CslH1 GACGCTCCACGGGATTC SEQ ID NO: 49 SJ85 CslH1 GGTTAGTTCCTTGTGCAGAGGTSEQ ID NO: 50 SJ91 CslH1 GAGCTGTGTTCGTGGAGCTTAG SEQ ID NO: 51 SJ163CslH1 CTGCTCTCGGCCACGGCCAT SEQ ID NO: 52 SJ164 CslH1CCGCCGGTTAGTTCCTTGTGCAGA SEQ ID NO: 53 SJ183 CslH1 GGAGAGTTCGTGTGCTGTGGSEQ ID NO: 54 SJ204 CslH1 CACCATGAGCCCCGTCCGGTTCGACA SEQ ID NO: 55 TUBAlpha tubulin CAAACCTCAGGGAAGCAGTCA SEQ ID NO: 56 TUB2F Alpha tubulinAGTGTCCTGTCCACCCACTC SEQ ID NO: 57 SJ107 CslF6 GCATCGTACTGGTGCTGCTSEQ ID NO: 58 SJ82 CslF6 GCGCTTCTCACGGGACACGTACA SEQ ID NO: 59 SJ94CslF4 GATGCGTACAACTCGAGCAA SEQ ID NO: 60 SJ95 CslF4 CGTTGCTGAAGTCAAGTGGASEQ ID NO: 61 SJ97 CslF9 CGCTGCAAACGAGAAAGAAGG SEQ ID NO: 62 SJ93 CslF9GGCGCTGAAATCCAGAGG SEQ ID NO: 63 SJ44 CslF3 CGGAAATCCATAGGAAAGGSEQ ID NO: 64 SJ38 CslF3 CGGCGGAACATGCAAC SEQ ID NO: 65 SJ96 CslF8GGATTGACCCAGCTGAAAAC SEQ ID NO: 66 SJ37 CslF8 GAGTTGTTGACGTAGTGGTCSEQ ID NO: 67 SJ244 Bx17 prom CGAGCACCCCAATCTACAGA SEQ ID NO: 68DNA, RNA Isolation and cDNA Synthesis

Plant DNA was isolated from fully expanded leaf tissue using a CTABbased method (Murray and Thompson, Nucleic Acids Res. 8: 4321-4325,1980). Total RNA was isolated from leaf and coleoptile tissues using anRNAeasy kit from Qiagen. RNA was isolated from developing endospermusing a phenol SDS method and LiCl precipitation (Clarke et al.,Functional and Integrative Genomics 8, 211-221, 2007). RNA was treatedwith DNAse using a “DNA-free” kit from Ambion and then cDNA wassynthesised using SuperscriptIII reverse transcriptase according to themanufacturer's instructions (Clontech).

Cloning of CslH Genes

The methods for cloning CslH genes were similar to those described inthe cloning and characterisation of CslF genes (Burton et al., PlantPhysiol 146: 1821-1833, 2008). A 1.8 kb tentative consensus sequence(TC140327) of a barley homolog of the rice Cellulose synthase like H1gene (LOC_Os10g20090) was identified in the TIGR database. PCR primerpairs (SJ27-SJ73 and SJ72-SJ75) were designed based on the rice CslH1sequence and used to amplify sequences from cDNA. The 5′ end of the genewas then amplified by 5′RACE using a SMART cDNA library and nested CslH1primers SJ28 and SJ79 according to the manufacturer's instructions(Clontech).

A full length genomic clone was isolated by amplification with primersSJ91 and SJ85 and Phusion Taq polymerase (Finnzymes) according to themanufacturers recommend cycling conditions (denature 30 sec at 98° C.followed by 35 cycles of 98° C. for 5 sec, 63° C. for 7 sec and 72° C.for 3 min) and cloned into the pCRBluntII TOPO cloning vector(Invitrogen).

Wheat CslH genomic clones were isolated by PCR with Phusion polymerasefrom the cultivar Chinese Spring using primers SJ163 and SJ164 and anannealing temperature of 70° C. A genome walking kit was used accordingto the manufacturers instructions (Clontech) to obtain sequencesextending upstream of the coding region of all three wheat CslHhomeologs from the variety Bob White (data not shown). A primer (SJ204)was designed that was specific to the third homeolog and used with SJ164to isolate the third full length genomic clone. It was confirmed thatthe predicted exon/intron boundaries could be spliced correctly bysequencing cDNA fragments (data not shown).

Expression Analysis of CslH Gene in Wheat and Barley by RT-PCR Total RNAwas isolated from sections of the first leaf of a 7 day old plant, fromdark grown coleoptiles of different ages, and from developing graincollected at 4 day intervals post anthesis (DPA), DNAse treated andreverse transcribed with Superscript III according to the manufacturer'sinstructions (Invitrogen). PCR reactions were performed using HotStarTaq(Qiagen). The cDNA was diluted and used in PCR reactions at a levelequivalent to 1 ng of original RNA per microlitre. For semi-quantitativeRT-PCR, CslH1 primers SJ72 and SJ74, for the CslF genes, primer pairswere as follows; (CslF6; SJ107-SJ82), (CslF4; SJ94-SJ95), (CslF9;SJ97-SJ93), (CslF3; SJ44-SJ38), (CslF8; SJ96-SJ37). An annealingtemperature of 59° C. was used. Test amplifications were performed toensure that the amplification was not saturated (approx 32-35 cyclesexcept tubulin 24 cycles) and the products were analysed by ethidiumbromide staining after agarose gel electrophoresis. Real time PCR wasperformed on triplicate samples on a Rotorgene 6000 machine (CorbettLife Sciences, AU) using HotStarTaq (Qiagen), SybrGreen and primersSJ183 and SJ164 and an annealing temperature of 60° C. Relativeexpression levels were calculated using the machine software with wheat0 dpa samples as the comparator (set to one). The Ct value of thissample was 25.5 cycles. For analysis of transgenic grain at 15 dpa, therelative expression values were normalised against tubulin and comparedto the lowest expression line (H1-13).

Expression Analysis of CslH Gene in Barley by Q-PCR

HvCslH1 transcript was measured in developing coleoptile 0.5 to 7 dayspost germination. HvCslH1 transcript was shown to accumulate only afterthe completion of the elongation phase and the emergence of the leaf.Highest levels of expression were seen at 7 days when the coleoptile issenescing (twisting and shrinking) (Gibeaut et al., Planta 221:729-738,2005).

Production of Transgenic Wheat Plants Overexpressing the Barley CslHGene in Endosperm

The full length barley cv. Himalaya genomic CslH sequence (SEQ ID NO:71) was amplified using primers SJ91 and SJ85, was inserted as an EcoRIfragment between a 1.9 kb fragment of the high molecular weight gluteninBx17 promoter and the nopaline synthase terminator (FIG. 23). The Bx17promoter confers high level expression in developing endosperm (Reddyand Appels, Theor Appl Genet 85: 616-624, 1993).

Bob White 26 wheat plants were transformed using the biolistics method(Pellegrineschi et al., Genome 45: 421-430, 2002) with 50 mg/L G418 asthe selection agent. The HvCslH expression vector (pZLBx17HvgH1 and asecond plasmid with the CaMV 35S promoter driving expression of theNPTII selectable marker (pCMSTLSneo, FIG. 24) were mixed in equimolaramounts and co bombarded into immature embryos.

Transgenic plants were screened for the presence of the transgene usingyoung leaf tissue and the RedExtractnAmp™ kit from Sigma with primersSJ244 and SJ79.

At anthesis (emergence of the anthers and shedding of pollen) heads weretagged to enable grain to be sampled at approximately 15 dpa. Threegrains from a head were pooled, RNA extracted and reverse transcribedand levels of transgene expression were analysed by real time PCR usingprimers SJ183 and SJ85. Expression levels were normalised against alphatubulin (primers TUB and TUB2F) and finally expressed as a ratiocompared to the lowest expresser.

Flour from mature single grains was analysed for beta glucan contentusing a scaled down version of the lichenase enzymatic method (AACCMethod 32-33, Megazyme assay kit, McCleary and Glennie-Holmes, J. InstBrewing 91: 285-295, 1985). Beta glucan contents are expressed as apercentage (w/w) of the milled whole grain flour.

EXAMPLE 13 Overexpression of the Barley CslH Gene in Barley Cv. GoldenPromise

The full-length coding region of the barley CslH cDNA (SEQ ID NO: 1) wastransferred into two Gateway-enabled barley transformation vectors. Thevector pRB474 contains the oat globulin promoter (Vickers et al., PlantMol Biol 62: 195-214, 2006) which provides endosperm specific expressionand the vector pMDC32 (Curtis and Grossniklaus, Plant Physiol. 133:462-9, 2003) contains a double 35S promoter which drives constitutiveexpression in all plant tissues.

Barley Transformation

The vectors were transferred into Agrobacterium tumefaciens and immaturescutella of the barley cultivar Golden Promise were transformed usingestablished protocols to produce two populations of transgenic plants.Insertion of the transgene was confirmed by Southern blotting. Plants236-1 to 236-18 contain the barley CslH gene driven by the oat globulinpromoter. Plants 237-1 and -2 contain the barley CslH gene driven by the35S promoter. Plants 208-2, -3, -5 and -7 are control plants and aretransgenic for the empty vector pRB474 carrying the oat globulinpromoter only.

Transcript Analysis

Leaf and developing grain samples, from 7 and 14 days after pollination(DAP) were collected from the 236 plants. Total RNA was extracted usingTRIzol reagent (Invitrogen) following a standard protocol and cDNA wassynthesized according to Burton et al., (Plant Physiol 146: 1821-1833,2008). Quantitative real-time PCR (QPCR) was carried out according toBurton et al. (2008, supra). The transcript levels of the CslH gene werecompared in the endosperm of the transgenic grain to wild type endospermlevels which are generally very low.

As shown in FIG. 26, the empty vector control lines (208) have typicalwild type levels of CslH transcript. The transgenic lines (236) showsignificantly increased HvCslH1 mRNA levels at 7 days (7 D) and furtherincreases at 14 days (14 D) after pollination.

Beta-Glucan Analysis

The T1 seed from the transgenic plants were collected. A sample of thebulked T1 grain from each individual plant was ground to flour and theamount of beta-glucan present was assayed using Megazyme method(described supra). The data from each plant are presented as the meanvalue of two replicates and the amount of beta-glucan as a percentage ofgrain weight is shown in Table 8, below:

TABLE 8 (1,3;1,4)-β-D-glucan content of bulked transgenic barley flour

The empty vector control lines (208) have a (1,3;1,4)-β-D-glucan contentaround 4% which is typical for wild type Golden Promise grain. Eventhough the T1 grain is bulked (and therefore contains null-segregantgrains) a significant number of the transgenic lines (shaded) show anoverall (1,3;1,4)-β-D-glucan content greater than the control, with thehighest value at 5.9%.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto, or indicated in this specification, individually or collectively,and any and all combinations of any two or more of the steps orfeatures.

Also, it must be noted that, as used herein, the singular forms “a”,“an” and “the” include plural aspects unless the context alreadydictates otherwise. Thus, for example, reference to “a transgene”includes a single transgene as well as two or more transgenes; “a plantcell” includes a single cell as well as two or more cells; and so forth.

What is claimed is:
 1. A method for modulating the level of(1,3;1,4)-β-D-glucan produced by a plant or fungal cell, the methodcomprising modulating the level and/or activity of a CslH-encoded(1,3;1,4)-β-D-glucan synthase in the cell, wherein the level and/oractivity of the a CslH-encoded (1,3;1,4)-β-D-glucan synthase ismodulated by modulating the expression of a CslH nucleic acid in thecell, and wherein modulating the expression of a CslH nucleic acid inthe cell results in modulation of the level of (1,3;1,4)-β-D-glucanproduced by the cell compared to a wild-type cell of the same taxon, andwherein the CslH nucleic acid comprises: (i) a nucleotide sequence setforth in SEQ ID NO: 72; (ii) a nucleotide sequence encoding the aminoacid sequence set forth in SEQ ID NO: 75; or (iii) a nucleotide sequenceencoding an amino acid sequence which is at least 50% identical to theamino acid sequence set forth in SEQ ID NO:
 75. 2. The method of claim 1wherein the cell is a plant cell.
 3. The method of claim 2 wherein thecell is a monocot plant cell.
 4. The method of claim 2 wherein the cellis a cereal crop plant cell.
 5. A plant or fungal cell comprising anyone or more of: a modulated level and/or activity of CslH-encoded(1,3;1,4)-β-D-glucan synthase relative to a wild type cell of the sametaxon; and/or modulated expression of a CslH nucleic acid relative to awild type cell of the same taxon, wherein the cell comprises a modulatedlevel of (1,3;1,4)-β-D-glucan relative to a wild-type cell of the sametaxon, wherein the CslH nucleic acid comprises: (i) a nucleotidesequence set forth in SEQ ID NO: 72; (ii) a nucleotide sequence encodingthe amino acid sequence set forth in SEQ ID NO: 75; or (iii) anucleotide sequence encoding an amino acid sequence which is at least50% identical to the amino acid sequence set forth in SEQ ID NO:
 75. 6.The cell of claim 5 wherein the cell is produced according to the methodof claim
 1. 7. The cell of claim 5 wherein the cell is a plant cell. 8.The cell of claim 7 wherein the cell is a monocot plant cell.
 9. Thecell of claim 7 wherein the cell is a cereal crop plant cell.
 10. Amulticellular structure comprising one or more cells according to claim5.
 11. The multicellular structure of claim 10 wherein the multicellularstructure is selected from the list consisting of a whole plant, a planttissue, a plant organ, a plant part, plant reproductive material orcultured plant tissue.
 12. The multicellular structure of claim 10wherein the multicellular structure comprises a cereal crop plant or atissue, organ or part thereof.
 13. The multicellular structure of claim12 wherein the multicellular structure comprises a cereal grain.
 14. Themulticellular structure of claim 11 wherein the multicellular structurecomprises a cell having modulated dietary fibre content relative to awild type cell of the same taxon.
 15. The multicellular structure ofclaim 14 wherein the multicellular structure comprises a cell having anincreased level of (1,3;1,4)-β-D-glucan relative to a wild type cell ofthe same taxon and an increased dietary fibre content relative to a wildtype cell of the same taxon.